ES2307568T3 - CPG TYPE NUCLEIC ACIDS AND SAME USE METHODS. - Google Patents

Abstract

Immunostimulatory compositions described as CpG-like nucleic acids are provided, including nucleic acids having immunostimulatory characteristics of CpG nucleic acid, despite certain substitutions of C, G, or C and G of the CpG dinucleotide. The substitutions can include, among others, exchange of methylated C for C, inosine for G, and ZpY for CpG, where Z is Cytosine or dSpacer and Y is inosine, 2-aminopurine, nebularine, or dSpacer. Also provided are methods for inducing an immune response in a subject using the CpG-like nucleic acids. The methods are useful in the treatment of a subject that has or is at risk of developing an infectious disease, allergy, asthma, cancer, anemia, thrombocytopenia, or nutropenia.

Description

CpG type nucleic acids and methods of using the same.

Related request

The application claims the priority of the US provisional application 60 / 254,341 filed on 8 December 2000

Field of the Invention

The present invention generally relates to immunostimulatory nucleic acids, to compositions of the themselves, and to methods of using nucleic acids immunostimulators

Background of the invention

Bacterial DNA, but not vertebrate DNA, has strong immunostimulatory effects for a wide variety of human and murine immune cells. Krieg AM et al. (1995) Nature 375: 546-9; Hartmann et al. (1999) Proc Natl Acad Sci USA 96: 9305-10; Hartmann G et al. (2000) J Immunol 164: 1617-24; Bauer M et al. (1999) Immunology 97: 699-705; Ballas ZK et al. (1996) J Immunol 157: 1840-5. Unmethylated CpG dinucleotides are less frequent in eukaryotic DNA than in bacterial DNA. Krieg AM et al. (1995) Nature 375: 546-9. It has been shown that unmethylated CpG dinucleotides within the context of specific flanking bases (called CpG motifs) have a wide variety of effects on human immune cells, such as B cells, natural killer cells (NK), T cells, macrophages, monocytes and dendritic cells. Parronchi et al. (1999) J immunol 163: 5946-53; Krieg AM (1999) Biochim Biophys Acta 1489: 107-16; Krieg AM et al. (1995) Nature 375: 546-9; Kranzer K et al. (2000) Immunology 99: 170-8; Hartmann G et al. (1999) Proc Natl Acad Sci USA 96: 9305-10; Hartmann G et al. (2000) J Immunol 164: 944-53; Hartmann G et al. (2000) J immunol 164: 1617-24.

As methylated vertebrate DNA is not immunostimulatory and it was known that methylated CpG oligonucleotides are not immune cell stimulators (Parronchi et al. (1999) J immunol 163: 5946-53; Hartmann G et al. (2000) J Immunol 164: 944-53), it was thought that methylation could, mainly, prevent immunostimulation by CpG-containing nucleic acids. For more applications in the field of antisense technology, where immunostimulation by the applied oligonucleotides is undesirable, many of the workers in the antisense field began using methylated oligonucleotides for their research with the specific purpose of avoiding unwanted immunostimulation.

In addition, while a number of studies show that C methylation of the CpG dinucleotide aborted Effectively the immunostimulatory effect of CpG, no study He has previously demonstrated the immunostimulatory effects of CpI.

Boggs, RT et al. (1997) Antisense & Nucleic Acid Drug Development vol. 7 pp 461-471 investigated the stimulatory effects of the CpG motif in specific sequence contexts, the effect of phosphorothioate and phosphodiester structures, methylating CpG cytosine and modifying all cytosines in an ODN.

Henry, S. Et al (2000). The Journal of Pharmacology and Experimental Therapeutics vol 292, No. 2, pp 468-479 investigated the immunostimulatory properties of oligodeoxynucleotide phosphorothioates in mice and the effect of chemical modifications such as the 5-methyl cytosine and 2'-methoxyethyl substituents.

Compendium of the invention

The invention provides compositions and methods useful for inducing an immune response and in treatment of allergy, asthma, cancer, infection, anemia, thrombocytopenia, and neutropenia The compositions are related to acids CpG nucleic acids called "CpG type nucleic acids" because they incorporate specific substitutions for C, G, or C and the G of the CpG dinucleotide while substantially retaining the immunostimulatory properties of acid molecules CpG nucleic.

The present invention provides a composition comprising an immunostimulatory nucleic acid that It has the sequence that includes at least the following formula:

5'X_ {1} X_ {2} CGX_ {3} X_ {4} 3 '

where C is a nucleotide where the nucleoside portion is 2'-methoxy citidine and where X 1, X 2, X 3 and X 4 are nucleotides, in a effective amount to induce an immune response. An acid immunostimulatory nucleic according to this embodiment of this aspect of the invention is referred to herein as a CpG nucleic acid 2'-methoxy-C or 2'-OMe-C or, equivalently, a CpG 2'-methoxy-C oligonucleotide or 2'-OMe-C. Also, according to this  aspect of the invention in some embodiments, the composition It also includes a pharmaceutically acceptable carrier and acid Nucleost immunostimulator is present in an amount effective for induce a response immune

In accordance with the preceding aspects and the Embodiments of the invention further apply the following. In some embodiments, the immunostimulatory nucleic acid is a isolated nucleic acid. In some embodiments, the nucleic acid immunostimulator has between 6 and 100 nucleotides, and in certain Preferred embodiments, between 8 and 40 nucleotides.

Also, according to embodiments preferred, the immunostimulatory nucleic acid has a structure  modified In some embodiments, the nucleic acid Immunostimulator is a synthetic nucleic acid.

In some embodiments, the nucleic acid immunostimulator is at least 18 nucleotides in length and is not an antisense nucleic acid.

The following also applies to compositions Pharmaceutical precedents of the invention that include an acid CpG type nucleic and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable vehicle It is a sustained release device.

Pharmaceutical compositions, in some embodiments, also include an antigen.

In some embodiments, the composition pharmaceutical also includes a medication anti-cancerous Preferably, the medication anti-cancer is selected from the group you sewed in a monoclonal antibody, a chemotherapeutic agent, and a radiotherapeutic agent

In some embodiments, the composition Pharmaceutical also includes an antiviral agent, an agent antibacterial, an antifungal agent, or an agent antiparasitic

In some embodiments, the composition Pharmaceutical also includes an ulcer medication.

In some embodiments, the composition Pharmaceutical also includes an allergy medication or a asthma medicine

In some embodiments, the composition Pharmaceutical also includes a medication for anemia, a medication for thrombocytopenia, or a medication for neutropenia

In some embodiments, the composition Pharmaceutical also includes a cytokine. Preferably, the cytokine is selected from the group consisting of interleukin-2 (IL-2), IL-3, IL-4, IL-18, interferon alfa (IFN-?), IFN-?, Tumor necrosis alpha factor (TNF-?), Flt3 ligand, stimulating factor of granulocyte colonies (G-CSF), and factor granulocyte-macrophage colony stimulant (GM-CSF).

In some embodiments, the composition Pharmaceutical also includes at least two nucleic acids immunostimulators that have different sequences.

In some embodiments, the composition Pharmaceutical also includes a CpG nucleic acid that has, at less, a non-methylated CpG motif.

Another aspect of the invention provides a method to induce an immune response. The method implies administration to an individual of a nucleic acid immunostimulator that has the sequence that includes at least the following formula:

5'X_ {1} X_ {2} CGX_ {3} X_ {4} 3 '

where C is a nucleotide where the nucleoside portion is 2'-methoxy citidine and where X 1, X 2, X 3 and X 4 are nucleotides, in a effective amount to induce a response immune

According to any of the methods preceding the invention, the following also applies. In some embodiments, the immunostimulatory nucleic acid is a isolated nucleic acid. In some embodiments, the nucleic acid immunostimulator has between 6 and 100 nucleotides, and in certain preferred aspects, between 8 and 40 nucleotides.

Also, according to embodiments preferred, the immunostimulatory nucleic acid has a structure  modified In more preferred embodiments, the structure modified in a modified phosphate structure.

In some embodiments, the individual will select from the group consisting of dog, cat, horse, cow, pig, sheep, goat, rabbit, guinea pig, non-human primate (for example, monkey), chicken, and fish (fish species, for example, salmon).

In some embodiments, the nucleic acid Immunostimulator is a synthetic nucleic acid.

According to some embodiments, the method It also includes administering an antigen. Preferably, the antigen is selected from the group consisting of an allergen, a tumor antigen, a viral antigen, a bacterial antigen, a fungal antigen, and a parasitic antigen. In some embodiments, the antigen is administered by mucosal route. Preferably, the mucosal route is selected from the group that It consists of oral, nasal, rectal, vaginal, transdermal, and ocular. In some embodiments, the antigen is administered by a route. parenteral Preferably, the parenteral route is selected from group consisting of intravenous, subcutaneous, intramuscular, and by direct injection.

In certain embodiments, the individual is in risk of developing an infectious disease and nucleic acid immunostimulator is administered in an effective amount to prevent Infectious disease

In certain embodiments, the individual has a Infectious disease and immunostimulatory nucleic acid is administered in an effective amount to treat the disease infectious

In certain embodiments, the individual is in risk of developing cancer and nucleic acid immunostimulator is administered in an amount effective to prevent or treat cancer.

In certain embodiments, the individual has or is at risk of developing an allergy and nucleic acid immunostimulator is administered in an amount effective to treat or prevent allergy

In some embodiments, the individual has or is at risk of developing asthma and nucleic acid immunostimulator is administered in an amount effective to treat or prevent asthma.

In some embodiments, the method in addition includes the administration of a therapy anti-cancer Preferably, the therapy anti-cancer is a monoclonal antibody specific for a tumor cell, chemotherapy, or a radiotherapy.

In some embodiments, the nucleic acid immunostimulator is given to an individual who has or is in risk of developing an immunodeficiency, in an effective amount to enhance spinal proliferation stimulation Bone in the individual. In some embodiments, the individual who have or are at risk of developing an immunodeficiency is a individual who suffers or is at risk of chemotherapy.

In some embodiments, the nucleic acid immunostimulator is given to an individual who has or is in risk of developing anemia, in an amount effective to potentiate Erythropoiesis in the individual.

In some embodiments, the nucleic acid immunostimulator is given to an individual who has or is in risk of developing thrombocytopenia, in an effective amount to enhance thrombopoiesis in the individual.

In some embodiments, the nucleic acid immunostimulator is given to an individual who has or is in risk of developing neutropenia, in an amount effective to enhance the proliferation of neutrophils in the individual.

In some embodiments, the nucleic acid immunostimulator is administered in an amount effective to induce Cytokine production. Preferably, the cytokine is select from the group consisting of IL-1β, IL-2, IL-6, IL-12, IL-18, TNF-?, IFN-?, And IFN-?.

In some embodiments, the nucleic acid immunostimulator is administered in an amount effective to Stimulate the activity of the natural killer cell.

In some embodiments, the nucleic acid Immunostimulator is administered by mucosal route. Preferably, the mucosal route is selected from the group that you sewed in oral, nasal, rectal, vaginal, transdermal and ocular.

In some embodiments, the nucleic acid Immunostimulator is administered by a parenteral route. Preferably, the parenteral route is selected from the group that sewed intravenously, subcutaneously, intramuscularly, and by injection direct.

In some embodiments, the nucleic acid immunostimulator is administered in a release vehicle sustained.

Brief description of the drawings

Figure 1 is a graph that represents the Human B cell activation by methylated and unmethylated CpG ODN.  Peripheral blood mononuclear cells (PBMC; 2x10 6 cells / ml) of a representative donor (n = 4) were incubated with 0.4 µg / ml, 1.0 µg / ml or 5.0 µg / ml oligodeoxynucleotide (ODN) 2006, 1758, 2186, or the corresponding 2117 methylated ODN, 1812 or 5107. Negative controls were incubated without ODN (w / o). The CD86 activation marker expression on CD19 + B cells It was measured by flow cytometry.

Figure 2 is a graph that represents the Human B cell activation by methylated and non-CpG ODN methylated. PBMCs from a representative donor (n = 3) were incubated with 0.4 µg / ml, 1.0 µg / ml or 10.0 µg / ml of the 2006 CpG ODN, the antisense CpG ODNs 5114 and 5116, or the corresponding ODNs methylated 5154 and 5155. Negative controls were incubated without ODN (w / o). B cell activation was analyzed by cytometry of flow, as described in Fig. 1.

Figure 3 on a graph representing the CD80 expression increase (left panel) and CD25 (panel right) on human B cells after incubation of PBMC with 0.4 µg / ml, 1.0 µg / ml or 10.0 µg / ml of ODN 2006, 2117 (2006 methylated) and 2137 (reason GpC 2006). The negative controls are incubated without ODN (w / o). The expression of the markers of B cell activation, CD80 and CD25, on CD19 + B cells determined by flow cytometry.

Figure 4 is a couple of graphics that represent the activation of human monocytes from peripheral blood by methylated ODN. PBMC (2x10 6 cells / ml) from donor 35 (left panel) and donor 24 (right panel) were incubated with 6 µg / ml ODN 2006, 2117, or 2137. Positive controls are incubated with 1 ng / ml of LPS, and the negative controls were incubated without ODN (w / o). CD80 expression on CD14 + monocytes It was determined by flow cytometry.

Figure 5 is a couple of graphics that represent the activation of human NK cells by ODP CpG methylated PBMC (2x10 6 cells / ml) from donors 24 (panel left) were incubated with 6 µg / ml ODN 2006, 2117, or 2137. As a positive control (for donor 24) PBMC were incubated with 100 U / ml of IL-2 together with 50 ng / ml of IL-12 To increase the expression of CD69 was added, with ODN, a submitogenic dose of IL-2 (10 U / ml). Negative controls were incubated without ODN (w / o). PBMC se stained for CD56 (N-CAM, NK cell marker), CD3 (T cell marker) and CD69 (early activation marker). The expression of CD69 in the population of CD56 + cells was measured by flow cytometry.

Figure 6 on a graph representing the activation of human natural T-killer cells (NKT) by ODN Methylated CpG. PBMC (2x10 6 cells / ml) were incubated with 6 ? / ml of ODN 2006, 2117, or 2137. The expression of CD69 over NKT CD56 + / CD3 + cells were analyzed by flow cytometry.

Figure 7 is a couple of graphics that represent the secretion of TNF-? e IL-6 by human PBCM after cultivation with ODN methylated and not methylated. PBMC (3x106 cells / ml) were incubated with ODN 2006, 2117, or 2137. Positive controls were incubated with 1 µg / ml lipopolysaccharide (LPS), and negative controls  they were incubated without ODN (w / o). The cytokines present in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). A. TNF-? Secretion (pg / ml) after incubation with 6 µg / ml ODN. B. Secretion of IL-6 (pg / ml) after incubation with 0.4 or 10.0 ODM / ml.

Figure 8 is a graph that represents the IL-1? secretion by human PBMC after culture with methylated and unmethylated ODN. PBMC (3x10 6 cells / ml) were incubated with 6 µg / ml of ODNs 2006, 2117, or 2137.  Positive controls were incubated with 1 µg / ml of LPS, and the Negative controls were incubated without ODN (w / o). It was measured by ELISA the IL-1? (pg / ml) present in the supernatants

Figure 9 is a couple of graphics that represent the secretion of IFN-? by PBMC humans after culturing them with methylated and unmethylated ODN. A. PBMC (5x10 6 cells / ml) were incubated with 6 µg / ml of ODNs 2006 or 2117. Positive controls were incubated with 0.1 µg / ml of LPS, and negative controls were incubated without ODN (w / o). It was measured by ELISA the IFN-? (pg / ml) present in the supernatants B. PBMC (5x106 cells / ml) were incubated with 6 ODMs 2006, 2117, or 2137, both with and without IL-2 (19 U / ml). The negative controls were cells incubated without ODN (w / o) and cells incubated only with IL-2 The ELISA was measured by IFN-? (Pg / ml) present in the supernatants

Figure 10 is a graph that represents the IL-10 secretion by human PBCM after incubation with methylated and unmethylated ODN. PBMC (5x106 cells / ml) is incubated with 6 µg / ml ODNs 2006, 2117, or 2137. Controls Negatives were incubated without ODN (w / o). The ELISA was measured by IL-10 (pg / ml) present in supernatants.

Figure 11 is a graph representing the activation of human B cell by the CpI ODN (G →) and by the ODN of modified cytosine structure 2'-methoxy (2'-OMe-C). PBMC (2x10 6 cells / ml) from donors 60 and 62 were incubated with 0.4 µg / ml, 1.0 µg / ml or 10.0 µg / ml of each ODN (2006, 1982, 5126, 5246, 5247, 5248, 5249, 5250 and 5251). Negative controls were incubated. without ODN (w / o). The expression of the activation marker CD86 on CD19 + B cells were measured by flow cytometry.

Figure 12 is a graph representing the B cell proliferation induced by ODP CpI (G →) and by the ODN of modified cytosine structure 2'-methoxy (2'-OMe-C). PBMC (2x10 6 cells / ml) of donors 48 and 62 were stained with the dye carboxyfluorescein succinimidyl diester diacetate (CFSE) and se incubated with 0.4 µg / ml, 1.0 µg / ml or 10.0 µg / ml of each ODN (2006, 1982, 5126, 5246, 5247, 5248, 5249, 5250 and 5251). The Negative controls were incubated without ODN (w / o). It was measured by flow cytometry the proliferation of B cells like the percentage of CD19 + B cells with decreasing brightness with dye CFSE

Figure 13 is a graph that represents the activation of human NK cells by the CpI ODN (G →) and by the ODN of modified cytosine structure 2'-methoxy (2'-OMe-C). PBMC (2x10 6 cells / ml) were incubated with 1.0 µg / ml or 6.0 µg / ml of each ODN Positive controls were incubated with IL-2 more IL-12 (IL-1 / IL-12), and controls Negatives were incubated without ODN (w / o). The expression of CD69 on NK CD56 + / CD3- cells were measured by flow cytometry. The control positive IL-2 / IL-12 for the Donor 41 activated 90% of CD69 positive NK cells.

Figure 14 is a graph that represents the Human monocyte activation of peripheral blood by ODP CpI (G \ rightarrowI) and by the modified structure ODN of the 2'-methoxy cytosine (2'-OMe-C). PBMC (2x10 6 cells / ml) from donors 67, 66, and 41 were incubated with 1.0 µg / ml and 6.0 µg / ml of each indicated ODN. The controls positive were incubated with 1 µg / ml of LPS, and controls Negatives were incubated without ODN (w / o). The cells were stained to CD14, CD19 and CD80 to measure flow cytometry expression of the CD80 cell surface marker on CD14 + monocytes excluding CD19 + / CD14 + B cells. The LPS activated 30% and 48% of Monocytes CD80 + from Donor 67 and Donor 66, respectively.

Figure 15 is a graph that represents the TNF-? secretion by human PBMC after cultivate them with the ODN CpI (G \ rightarrowI) and by the ODN of modified structure of cytosine 2'-methoxy (2'-OMe-C). PBMC (5x10 6 cells / ml) from donors 94 and 95 were incubated with 6.0 µg / ml of the indicated ODN or of LPS (0.1 µg / ml). Negative controls they were incubated without ODN or LPS (w / o). The ELISA was measured by TNF-? Present in the supernatants of cell culture (pg / ml). LPS induced 677 pg / ml and 1000 pg / ml of TNF-? For the PMBCs of Donors 95 and 94, respectively.

Figure 16 is a graph representing the IFN-? secretion by human PMBC after cultivate them with the ODN CpI (G \ rightarrowI) and by the ODN of modified structure of cytosine 2'-methoxy (2'-OMe-C). PBMC (5x10 6 cells / ml) from donors 63 and 61 were incubated with 6.0 µg / ml of the indicated ODN. Negative controls were incubated without ODN (w / o). The IFN-γ of the cell culture supernatants (pg / ml).

Figure 17 is a graph that represents the IL-10 secretion by human PBMC after incubation with the CpI ODN (G \ rightarrowI) and by the structure ODN modified cytosine 2'-methoxy (2'-OMe-C). PBMC (5x10 6 cells / ml) from donors 63 and 61 were incubated with 6.0 µg / ml of the indicated ODN. Negative controls were incubated without ODN (w / o). The IL-10 of the cell culture supernatants (pg / ml).

Figure 18 is a graph that represents the IL-6 secretion by human PBMC after incubation with the CpI ODN (G \ rightarrowI) and by the structure ODN modified cytosine 2'-methoxy (2'-OMe-C). PBMC (5x10 6 cells / ml) from donors 95 and 94 were incubated with 6.0 µg / ml of the indicated ODN. Negative controls were incubated without ODN (w / o). The IL-6 of the cell culture supernatants (pg / ml).

Detailed description

According to the invention, it was discovered in a manner surprising that certain CpG type nucleic acids are acids immunostimulatory nuclei despite their lack of dinucleotides Nonmethylated CpG, which have previously been shown to be crucial in the immunostimulatory effects of CpG nucleic acids. The CpG type nucleic acids are useful in any application where nucleic acids are effective in inducing a response biological activator, stimulant, or proliferative.

The unmethylated CpG sequences, although they are relatively rare in humans, they are commonly found in the DNA of infectious organisms such as bacteria. The system human immune system apparently has developed to recognize unmethylated CpG sequences as a clear signal of infection warning and to initiate an immune response potent and immediate against invading pathogens. In this way, the nucleic acids containing unmethylated CpG, depending on this Innate immune defense mechanism, can use a unique route and natural for immune therapy without causing adverse reactions seen frequently with other immune stimulating agents. The effects of unmethylated CpG nucleic acids on immune modulation have been extensively described in patent applications published, such as PCT / US95 / 01570; PCT / US97 / 19791; PCT / US98 / 03678; PCT / US98 / 10408; PCT / US98 / 04703; PCT / US99 / 07335; Y PCT / US99 / 09863.

A "CpG nucleic acid" is a nucleic acid that includes at least one unmethylated CpG dinucleotide. A nucleic acid that contains at least one unmethylated CpG dinucleotide is a nucleic acid molecule that contains an unmethylated cytosine in a cytosine-guanine dinucleotide sequence (ie, "CpG DNA" or DNA containing a 5'cytosine followed by 3 'guanine and linked by a phosphate bond) and that activates the immune system. CpG nucleic acids can be double stranded or single stranded. Generally, double chain molecules are more stable in vivo , while single chain molecules have increased immune activity. Therefore, in some aspects of the invention, it is preferred that the nucleic acid be single stranded and in other aspects it is preferred that the nucleic acid be double stranded. The terms CpG nucleic acid or CpG oligonucleotide used herein refer to an immunostimulatory CpG nucleic acid or an immunostimulatory CpG oliginucleotide unless otherwise indicated. The entire immunostimulatory CpG nucleic acid may be unmethylated or there may be unmethylated portions, but at least the C of dinucleotide 5'-CG-3 'must be unmethylated.

An "immunostimulatory nucleic acid", such as used here, is any nucleic acid that contains a immunostimulatory motif or a structure that induces a response immune An immune response, as used here, includes stimulation of immune cells and non-immune cells to secrete or express factors that participate in and / or characterize the immune activation Therefore, this term includes, without limitations, stimulation of cytokine secretion by several cell types, including lymphocytes, professional cells antigen presenters (APCs, which include dendritic cells), and epithelial cells; secretion stimulation of immunoglobulins by B cells; and expression stimulation of cell surface molecules of molecules co-stimulators and co-receptors on T cells, B cells, natural killer cells (NK), monocytes, macrophages, and APCs.

An effective amount of nucleic acid immunostimulator to induce an immune response is a amount of immunostimulatory nucleic acid that, when administer to an individual or contact cells of a individual, induces stimulation of immune cells and / or non-cells immune to secrete or express factors that participate in and / or characterize an immune activation. In certain embodiments, the Amount is effective to induce cytokine secretion. Examples of cytokines are given below. They are well known in the state of the technique methods to determine the induction and secretion of cytokines, and include enzyme-linked immunosorbent assays (ELISA), intracellular cellular classification activated by fluorescence (FACS), and bioassays. In certain other embodiments, The amount is effective to stimulate the activity of NK cells. The methods to determine the activity of NK cells are fine known in the state of the art and include the determination of target cell death (cell lysis), marker expression of CD69 activation on the cell surface (for example, by FACS) and gamma interferon secretion (IFN-?; by example, by ELISA). Additional examples of specific markers cell surface immune activation may include without limitations, the expression of CD11b, CD25, CD28, CD43, CD54, CD62L, CD71, CD80, CD86, CD95L, CD106, CD134, and CD134L.

The terms "nucleic acid" and "oligonucleotide" are used interchangeably to denote multiple nucleotides (i.e. molecules that comprise a sugar (for example, ribose or deoxyribose) bound to a group phosphate and a variable organic base, which is both a pyrimidine substituted (for example, cytosine (C), thymine (T), or uracil (U)) or a substituted purine (for example, adenine (A) or guanine (G)). C derivatives are also included, including without limitation 2 ', 3'-dideoxycytidine (ddC), 5'-bromo-ddC, 3'-amino-2,3-ddC, 5-iodo-2'-deoxicitidine (dC), and derivatives of G, which include without limitations 3'-azido-2 ', 3'-dideoxyguanosine  (ddG), 7-deaza-2'-deoxiguanosine (dG), 8-bromo-dG, 8-bromo-2'-dG, O6-methyl-2'-dG. Such as used here, the terms "nucleic acid" and "oligonucleotide" refers to oligoribonucleotides as well as oligodeoxyribonucleotides (ODN). The terms could also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic polymer that contains a base. Nucleic acids include vectors, for example, plasmids, as well. as oligonucleotides. Nucleic acid molecules can be obtain from natural sources of nucleic acids (for example, DNA genomic or cDNA of prokaryotes that include bacteria and of eukaryotes that include washes) and are referred to here as "isolated", but are preferably synthetic (for example, produced by oligonucleotide synthesis).

In one aspect, the invention provides an acid. CpG type nucleic. As used here, an "acid CpG type nucleic "refers to a nucleic acid immunostimulator that has all the characteristics of an acid CpG nucleic as described herein, with at least one of the following exceptions: (1) the C cytosine base of at least one CpG dinucleotide is 5'methylated (methylated CpG); (2) the G of, at less, a CpG dinucleotide is substituted by an I (inosine; CpI nucleic acid); (3) C sugar of at least one CpG dinucleotide is modified to be a 2'-alkoxy cytosine; (4) The C of at least one CpG dinucleotide is substituted by Z, where Z is selected from 2'-deoxyuridine (dU), 5-fluoro-2'-dU, and dSpacer, where dSpacer is a serving of sugar without a base; (5) the G of at least one CpG dinucleotide is substituted by Y, where And it is selected from 2-aminopurine, xanthosine, N7-methyl-xanthosine, nebularin, and dSpacer, where dSpacer is a sugar position without a base. From according to certain preferred embodiments, the 2'-alkoxy cytosine is 2'-methoxy cytosine (nucleic acid 2'-OMe-cytosine).

In this way, CpG type nucleic acids may incorporate certain modifications or substitutions that they involve both the bases and the sugar portions of the structure. Those with methylated CpG involve, in embodiments preferred, the 5 'methylation of the cytosine base. Those with CpI involve the use of a particular base, inosine Those with 2'-OMe-cytosine involve the substitution of a methoxy group (OCH3; OMe) with a hydroxy group (OH) in position 2 'of the sugar portion attached to the base cytosine

In some embodiments, a nucleic acid of CpG type may be based on a CpG nucleic acid previously known. In other aspects, a CpG type nucleic acid can be different from a previously known CpG nucleic acid. Too, according to some preferred aspects, the nucleic acid of CpG type has a modified structure, which includes a modified phosphate structure (see below).

According to a preferred embodiment of the invention, the CpG type nucleic acid is at least 18 nucleotides in length and is not an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid sequence that (1) has both a sequence complementarity with a structural gene sequence of the host, or can be specifically hybridized, under strict conditions, with a Nucleic acid sequence of the treated host, and (2) when complexed with the structural gene or nucleic acid sequence, causes a loss of function of the structural gene or nucleic acid sequence. An antisense nucleic acid specifically hybridizes when the binding of the compound to the target DNA or the RNA molecule interferes with the normal function of the DNA or the target RNA to cause a loss of utility, and there is a sufficient degree of complementarity to prevent non-specific binding. of the antisense compound to non-target sequences under conditions where specific binding is desired, that is, under physiological conditions in the case of in vivo tests or in a therapeutic treatment, and in the case of in vitro tests, under conditions in which They perform the tests. The mere complementarity to chromosomal DNA or messenger RNA should not define antisense, because not all complementary sequences with a particular gene or nucleic acid sequence cause a loss of function of the particular gene or nucleic acid sequence. Typically, an antisense nucleic acid will be able to hybridize, under strict conditions, with a sequence or correspond to the coding region of a gene, including and, particularly, involving an intron. Alternatively, an antisense nucleic acid will typically be able to hybridize, under strict conditions, with a sequence in a regulatory element associated with a gene, for example, a promoter or enhancer. Those skilled in the art can determine, without undue experimentation, whether a CpG type nucleic acid is an antisense nucleic acid.

As used herein, a "nucleic acid Methylated CpG "refers to an immunostimulatory nucleic acid which has all the characteristics of a CpG nucleic acid such as described here, with the exception that the C of at least a CpG dinucleotide is methylated. This discovery was quite unexpected. It is widely demonstrated that when the C of a CpG dinucleotide is replaced by a 5-methylcytosine, activity is lost immunostimulatory Therefore, surprisingly, it was found that the replacement of the C of a CpG dinucleotide by 5-methylcytosine did not cause this profound loss of activity. Said immunostimulatory nucleic acid may include, at less, a methylated CpG dinucleotide and at least one CpG dinucleotide not methylated In an alternative aspect, the nucleotide acid Whole immunostimulator may be methylated, including all CpG dinucleotides present.

One aspect of this revelation provides a immunostimulatory nucleic acid which is a CpG nucleic acid methylated represented by at least the formula:

5'X_ {1} X_ {2} CGX_ {3} X_ {4} 3 '

where C is methylated and X_ {1}, X2, X3 and X4 are nucleotides. In one version, X_ {2} It is adenine, guanine, cytosine, or thymidine. In another version, X_ {3} It is adenine, guanine, cytosine, or thymidine. In another version, X_ {2} it is guanine, adenine, or thymine and X3 is cytosine, adenine, or thymine

In another version, the nucleic acid immunostimulator in an isolated methylated CpG nucleic acid represented by at least the formula:

5'N_ {1} X_ {1} X_ {2} CGX_ {3} X_ {4} N_ {3}

where C is methylated and X_ {1}, X 2, X 3 and X 4 are nucleotides, and N 1 and N 2 are nucleic acid sequences composed of about 0-25 nucleotides each. In one version, X_ {1} X_ {2} is a dinucleotide selected from the group that It consists of: GpG, GpA, GpT, ApG, ApA, ApT, CpG, CpA, CpT, TpG, TpA, and TpT; and X 3 X 4 is a dinucleotide selected from the group consisting of: ApG, ApA, ApT, ApC, CpG, CpA, CpC, TpG, TpA, TpT, and TpC Preferably, X 1 X 2 is GpA or GpT and X 3 X 4 It is TpT. In other versions, X_ {1} or X_ {2} or both are purines and X 3 or X 4 or both are pyrimidines or X 1 X 2 is GpA and X 3 or X 4 or both are pyrimidines. In another version, X_ {1} X_ {2} is a dinucleotide selected from the group that It consists of: TpA, ApA, ApC, ApG, and GpG. In another version, X_ {3} X_ {4} is a dinucleotide selected from the group that It consists of: ApG, ApA, ApC, TpG, TpA, TpT, and CpA. X_ {1} X_ {2}, in another version is a dinucleotide selected from the group that It consists of: GpT, GpC, ApT, TpG, TpT, TpC, CpG, CpT, and CpC

In another version, the nucleic acid immunostimulator has the sequence

5'TCNTX_ {1} X_ {2} CGX_ {3} X_ {4} 3 '

where the C of the dinucleotide CG is methylated, and X1, X2, X3 and X4 are nucleotides, and N it is a nucleic acid sequence composed of about 0-25 nucleotides Nucleic acid revelation immunostimulator, in some versions, includes X_ {1} X_ {2} selected from the group you sewed in GpG, GpA, GpT, and ApA and X_ {3} X_ {4} is selected from the group consisting of TpT, TpC, and CpT

As used herein, a "CpI nucleic acid" refers to an immunostimulatory nucleic acid that has all the characteristics of a CpG nucleic acid as described herein, with the exception that the G of at least one dinucleotide CpG is replaced by an inosine (I). Inosine is a purine that represents a deaminated form of adenosine or guanosine in vertebrates. Atryer L, Biochemistry , 4th ed., WH Freeman, 1995. In accordance with this aspect of the invention, it has been found that ODN phosphorothioate with guanosine changes by inosine are as effective as immunostimulatory nucleic acids such as CpG phosphorothioate ODN nucleic acids. This discovery was quite unexpected. Surprisingly, replacing the G of the CpG dinucleotide with another purine, I, did not cause a loss of activity. Said immunostimulatory nucleic acid may include at least one CpI dinucleotide and at least one methylated or non-methylated CpG dinucleotide. In an alternative version, the immunostimulatory nucleic acid may have the same sequence as a CpG nucleic acid except that all CpG dinucleotides are substituted by CpI dinucleotides. In a more alternative version, the immunostimulatory nucleic acid may have the same sequence as a CpG nucleic acid except that all guanosine bases are substituted by inosine bases.

In one version, a nucleic acid is provided immunostimulator which is a CpI nucleic acid represented by, at less, the formula:

5'X_ {1} X_ {2} CIX_ {3} X_ {4} 3 '

where C is cytosine, I is inosine, and X_ {1}, X_ {2}, X_ {3}. In certain versions, the C of CpI dinucleotide of the above formula is not methylated. In others certain versions, the CpI dinucleotide C of the formula above  It is methylated. In one version, X_ {2} is adenine, guanine, inosine, cytosine, or thymine. In another version, X_ {3} is adenine, guanine, inosine, cytosine, or thymine. In another version, X_ {2} is guanine, adenine, or thymine and X3 is cytosine, adenine, or thymine

In another version, the nucleic acid immunostimulator is an isolated CpI nucleic acid represented by, at least the formula:

5'N_ {1} X_ {1} X_ {2} CIX_ {3} X_ {4} N_ {2} 3 '

where C is cytosine, I is inosine, and X1, X2, X3 and X4 are nucleotides, and N1 and N2 are nucleic acid sequences composed of about of 0-25 nucleotides each. In certain versions, C of the CpI dinucleotide of the above formula is not methylated. In one version, X_ {1} X_ {2} is a dinucleotide selected from the group consisting of: GpG, GpA, GpT, ApG, IpI, IpT, GpI, IpA, pT, ApI, ApA, ApT, CpG, CpI, CpA, CpT, TpG, TpI, TpA, and TpT; Y X_ {3} X_ {4} is a dinucleotide selected from the group that It consists of: ApG, ApI, ApA, ApT, ApC, CpG, CpI, CpA, CpC, TpG, TpI, TpA, TpT, and TpC. Alternatively, X_ {2} is GpA, GpT, IpA, or IpT, and X_ {3} X_ {4} is TpT. In other versions, X_ {1} or X_ {2} or both are purines and X 3 or X 4 or both are pyrimidines, or X_ {1} X_ {2} is GpA or IpA and X_ {3} or X_ {4} or both are pyrimidines In another version, X_ {1} X_ {2} is a dinucleotide selected from the group consisting of: TpA, ApA, ApC, ApG, ApI, IpI,  IpG, GpI, and GpG. In another version, X_ {3} X_ {4} is a Dinucleotide selected from the group consisting of: ApG, ApI, ApA, ApC, TpG, TpI, TpA, TpT, and CpA. In another version, X_ {1} X_ {2} is a dinucleotide selected from the group consisting of: GpT, GpC, IpT, OpC, ApT, TpG, TpI, TpT, TpC, CpG, CpI, CpT, and CpC

In another version, the nucleic acid immunostimulator has the sequence

5'TCNTX_ {1} X_ {2} CIX_ {3} X_ {4} 3 '

where C is cytosine, I is inosine, X 1, X 2, X 3 and X 4 are nucleotides, and N is a nucleic acid sequence composed of about 0-25 nucleotides In certain versions, the C of CpI dinucleotide of the above formula is not methylated. The immunostimulatory nucleic acids of this disclosure, in some versions, include X_ {1} X_ {2} selected from the group that sewed on GpG, GpA, GpT, IpI, IpA, IpT, IpG, GpI, and ApA and X_ {3} X_ {4} is selected from the group consisting of TpT, TpC, and CpT

As used herein, a "nucleic acid CpG 2'-alkoxy cytosine "refers to an acid immunostimulatory nucleic that has all the characteristics of a CpG nucleic acid as described herein, with the exception of  that the sugar portion of the C of at least one CpG dinucleotide is rented in position 2 ', thus, replacing a C with a 2'-O-alkyl cytosine. In a preferred embodiment, the sugar portion of the C of at least a CpG dinucleotide is methylated at position 2 ', thus, replacing a C with a 2'-O-methyl cytosine (2'-OMe C). Said CpG quil nucleic acids cytosine may include at least one CpG dinucleotide O-alkyl cytosine and at least one CpG dinucleotide not modified. In an alternative embodiment, all cytosine of the acid CpG type nucleic may be a 2'-alkoxy cytosine, including all CpG dinucleotides present.

The discovery that the CpG 2'-alkoxy cytosine nucleic acids of the invention are immunostimulators was quite unexpected. Previous studies showed that the substitution of a specific individual deoxynucleoside or pairs of deoxynucleosides flanking a CpG dinucleotide with the corresponding 2'-methoxy deoxynucleosides affects the immunostimulatory potential of the oligonucleotide sequence in a highly position dependent manner. Zhao Q et al. (1999) Bioorg Med Chem Lett 9: 3453-8; Zhao Q et al. (2000) Bioorg Med Chem Lett 10: 1051-4. According to these demonstrations, the closer to the CpG dinucleotide the nucleoside substitution is made, the weaker the immunostimulatory potential of the oligonucleotide as a whole. Surprisingly, it was found, according to the immediate invention, that the substitution of cytosine with 2'-alkoxy cytosine had no dramatic influence on the immunostimulatory activity of the oligonucleotide.

In certain preferred embodiments, the invention provides an immunostimulatory nucleic acid that is a CpG 2'-alkoxy cytosine nucleic acid represented for at least the formula:

5'X_ {1} X_ {2} CGX_ {3} X_ {4} 3 '

where C is a nucleotide in which The nucleoside portion is 2'-alkoxy cytosine. In a aspect, X2 is adenine, guanine, cytosine, or thymine. In other aspect, X 3 is adenine, guanine, cytosine, or thymine. In other aspect, X2 is guanine, adenine, or thymine and X3 is cytosine, adenine, or thymine

In other embodiments, the nucleic acid immunostimulator is a CpG 2'-alkoxy nucleic acid Isolated cytosine represented by at least the formula:

5'N_ {1} X_ {1} X_ {2} CGX_ {3} X_ {4} N_ {3}

where C is 2'-alkoxy cytosine, X1, X2, X3 and X 4 are nucleotides, and N 1 and N 2 are sequences of nucleic acids composed of about 0-25 nucleotides each. In one embodiment, X_ {1} X_ {2} is a Dinucleotide selected from the group consisting of: GpG, GpA, GpT, ApG, ApA, ApT, CpG, CpA, CpT, TpG, TpA, and TpT; and X_ {3} X_ {4} is a dinucleotide selected from the group consisting of: ApG, ApA, ApT, ApC, CpG, CpA, CpC, TpG, TpA, TpT, and TpC. Preferably, X_ {1} X_ {2} is GpA or GpT and X_ {3} X_ {4} is TpT. In others embodiments, X 1 or X 2 or both are purines and X 3 or X 4 or both are pyrimidines or X 1 X 2 is GpA and X 3 or X4 or both are pyrimidines. In another preferred embodiment, X_ {1} X_ {2} is a dinucleotide selected from the group that It consists of: TpA, ApA, ApC, ApG, and GpG. In another embodiment, X_ {3} X_ {4} is a dinucleotide selected from the group that It consists of: ApG, ApA, ApC, TpG, TpA, TpT, and CpA. X_ {1} X_ {2}, in another embodiment is a dinucleotide selected from the group that It consists of: GpT, GpC, ApT, TpG, TpT, TpC, CpG, CpT, and CpC

In another preferred embodiment, the nucleic acid immunostimulator has the sequence

5'TCNTX_ {1} X_ {2} CGX_ {3} X_ {4} 3 '

where the C of the CG dinucleotide is 2'-alkoxy cytosine, X1, X2, X3 and X4 are nucleotides, and N is a nucleic acid sequence composed of about 0-25 nucleotides. The immunostimulatory nucleic acids of the invention, in some versions, include X_ {1} X_ {2} selected from the group that sewn in GpG, GpA, GpT, and ApA and X_ {3} X_ {4} is selected from group consisting of TpT, TpC, and CpT

In other more remote embodiments, the invention provides CpG type nucleic acids that incorporate a combination of the types of modifications listed above. Therefore, the invention also provides nucleic acids of the type CpG incorporating a CpI dinucleotide and, or a methylated C in the CpI dinucleotide or a 2'-alkoxy cytosine in the CpI dinucleotide. Likewise, the invention also encompasses acids CpG-type nuclei that incorporate both methylated cytosine in CpG dinucleotide and a 2'-alkoxy cytosine in the CpI dinucleotide.

Another aspect of the disclosure provides acids CpG type immunostimulatory nuclei that have the sequence which includes at least the formula

5'X_ {1} X_ {2} ZYX_ {3} X_ {4} 3 '

where Z is selected from the group that It consists of cytosine and dSpacer, and is selected from the group that it consists of inosine, 2-aminopurine, nebularin, and dSpacer; Z is not cytosine when Y is inosine; and X_ {1}, X_ {2}, X 3 and X 4 are nucleotides. Such nucleic acids CpG type immunostimulators are referred to herein as nucleotides ZpY.

The term "dSpacer", as used here, it refers to a sugar-phosphate portion that is as a nucleotide lacking a base. This entity can be incorporated into the structure of a nucleic acid by junctions  5'-a-3'binding nucleotides.

In versions where Z is cytosine, it may be methylated or not methylated.

In some versions, according to this aspect of the invention, the immunostimulatory nucleic acid has the sequence that includes at least the formula

5'TCNTX_ {1} X_ {2} ZYX_ {3} X_ {4} 3 '

where N is an acid sequence nuclei composed of about 0-25 nucleotides

In some versions, according to this aspect of the disclosure, the immunostimulatory nucleic acid is a isolated nucleic acid.

It is some embodiments, the invention provides a pharmaceutical composition that includes at least one effective amount of the immunostimulatory nucleic acid to induce an immune response and a pharmaceutically vehicle acceptable. The pharmaceutical composition according to this embodiment of the invention, can be prepared by placing an effective amount of the immunostimulatory nucleic acid to induce a response immune in a pharmaceutically acceptable vehicle. The vehicle Pharmaceutically acceptable may, in some embodiments, include a sustained release device. Other medications and Agents can be included in the pharmaceutical composition, including an antigen, an antibiotic (antiviral agents, antibacterial, antifungal, and antiparasitic), a medication for the ulcer, an allergy or asthma medication, a medication for the treatment of anemia, thrombocytopenia, or neutropenia, and a cytokine In certain preferred embodiments that include a cytokine, the cytokine is selected from IL-2,  IL-3, IL-4, IL-18, IFN-?, IFN-?, Ligand of Flt3, G-CSF, and GM-CSF.

Pharmaceutical compositions containing a immunostimulatory nucleic acid according to this aspect of the invention, will include, in some embodiments, a plurality of immunostimulatory nucleic acids that have sequences different. In some embodiments, the pharmaceutical composition may include a CpG nucleic acid that has at least one reason CpG not methylated.

In certain preferred embodiments, the acid Nucleost immunostimulator has between 6 and 100 nucleotides, preferably enter 8 and 40 nucleotides.

Also, according to this aspect of the invention, in some embodiments where the nucleic acid Immunostimulator has a modified structure. In certain preferred embodiments, the modified structure is a structure  modified phosphate.

To facilitate incorporation into cells, the immunostimulatory nucleic acids are preferably of a range from 6 to 100 bases in length. However, nucleic acids from any size larger than 6 nucleotides (even many kb of length) are able to induce an immune response of according to the invention if sufficient motives are present immunostimulators Preferably, the CpG type nucleic acid it is in the size range of between 8 and 100 and in some embodiments, between 8 and 40 or between 8 and 30 nucleotides of size.

CpG type nucleic acids can be formulated together with at least one other nucleic acid. In some cases, the at least one other nucleic acid is a nucleic acid immunostimulator, for example, a CpG nucleic acid or other acid CpG type nucleic. In other cases, the at least one other acid Nucleic is a vector nucleic acid.

CpG type nucleic acids can be co-administered with at least one other nucleic acid. In In some cases, the at least one other nucleic acid is a nucleic acid. immunostimulator, for example, a CpG nucleic acid or other acid CpG type nucleic. In other cases, the at least one other acid Nucleic is a vector nucleic acid.

In the case that the nucleic acid immunostimulator be co-administered with an acid Nucleic vector, under certain circumstances, is useful if the Immunostimulatory nucleic acid structure is a combination chimeric phosphodiester and phosphorothioate (or other modification phosphate). A cell may have difficulty incorporating a plasmid vector in the presence of a completely oligonucleotide phosphorothioate Therefore, when both a vector and a oligonucleotide, are administered to an individual, it is preferred that the oligonucleotide has a chimeric structure, or, if the oligonucleotide has a completely phosphorothioate structure, that the plasmid is associated with the vehicle that administers it directly inside the cell, thereby preventing the need Cellular absorption Such vehicles are known in the state of the technique, for example, liposomes and gene guns.

In the case that more than one acid is administered immunostimulatory nucleic, either alone or in conjunction with a vector, the structure of an immunostimulatory nucleic acid can be completely phosphorothioate and the structure of another acid Fully phosphodiester immunostimulatory nucleic. Therefore, for example, an ODN phosphorothioate can be together with an ODN phosphodiester

For use in the invention, immunostimulatory nucleic acids can be synthesized de novo using any number of procedures known in the state of the art. Such compounds are referred to as "synthetic nucleic acids." These synthesis methods include, for example, the β-cyanoethyl phosphoramidite method (Beaucage SL and Caruthers MH (1981) Tetrahedron Lett 22: 1859), and the nucleoside H-phosphonate method (Garegg et al. (1986) Tetrahedron Lett 27 : 4051-4054; Froehler et al. (1986) Nucl Acid Res 14: 5399-5407; Garegg et al. (1986) Tetrahedron Lett 27: 4055-4058; Gaffney et al. (1988) Tetrahedron Lett 29: 2619-2622 ). These chemical reactions can be performed by a variety of commercially available automatic nucleotide synthesizers. These nucleic acids refer to synthetic nucleic acids. Alternatively, immunostimulatory nucleic acids can be produced on a large scale in plasmids (see Sanbrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, New York, 1989) and can be separated into smaller pieces or be administered whole. Nucleic acids can be prepared from natural nucleic acid sequences (eg, genomic DNA or cDNA) using known techniques, such as those that employ restriction enzymes, exonucleases or endinucleases. Nucleic acids prepared in this way are called isolated nucleic acids. The term "immunostimulatory nucleic acid" comprises both stimulatory, synthetic and isolated nucleic acids.

For use in vivo , nucleic acids are preferably relatively resistant to degradation (for example, they are stable). A "stable nucleic acid molecule" would mean a nucleic acid molecule that is relatively resistant to degradation in vivo (for example, via an exo- or endo nuclease). Stabilization can be a function of secondary length or structure. Immunostimulatory nucleic acids that are tens to hundreds of kb in length are relatively resistant to degradation in vivo. For shorter immunostimulatory nucleic acids, the secondary structure can stabilize and increase its effect. For example, if the 3 'end of a nucleic acid has self-complementarity with an upstream region, so that it can fold back and form a type of stem-loop structure, then the nucleic acid becomes stable and thus shows more activity

Alternatively, the stabilization of a nucleic acid can be achieved via modifications of the structure.

Preferred stabilized nucleic acids of the invention have modified structure. Modification of the nucleic acid structure has been shown to provide enhanced activity of immunostimulatory nucleic acids when administered in vivo . One type of modified structure is a modification of phosphate structure. The inclusion, in immunostimulatory nucleic acids, of at least two phosphorothioate bonds at or near the 5 'end of the oligonucleotide and multiple (preferably five) phosphorothioate bonds at or near the 3' end, in some circumstances, can provide maximum activity and protect the nucleic acid from degradation by exo- and extracellular endonucleases. Other modified phosphate nucleic acids include modified phosphodiester nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, alkyl phosphonate and aryl phosphonate, alkyl phosphorothioate and aryl phosphorothioate, phosphorodithinate, and combinations thereof. Each of these combinations in CpG nucleic acids and their particular effects on immune cells is discussed in more detail in published patent applications PCT / US95 / 01570 and PCT / US97 / 19791.

Although Applicants are not bound by the theory, it is believed that these modified phosphate nucleic acids may show more stimulatory activity due to enhanced nuclease resistance, increased cellular absorption, increased of protein binding, and / or altered intracellular localization.

Modified structures such as phosphorothioates can be synthesized using automated techniques that employ both phosphoramidate and H-phosphonate chemical compounds as described above. The aryl- and alkyl phosphonates can be made, for example, as described in US Patent No. 4,469,863; and alkyl phosphotriesters (in which the portion of charged oxygen is alkylated as described in US Patent No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other modifications and substitutions in the structure of the DNA have been described. Uhlmann E and Peyman A (1990) Chem Rev 90: 544; Goodchild J (1990) Bioconjugate Chem 1: 165.

Both phosphorothioate and phosphodiester nucleic acids that contain immunostimulatory motifs are active in immune cells. However, based on the concentration necessary to induce the stimulatory effects of the specific nucleic acid, the immunostimulatory nucleic acids of nuclease-resistant phosphorothioate structure are more potent. In certain in vitro assays , CpG phosphorothioate is 2 orders of magnitude more potent than CpG phosphodiester. Krieg AM et al. (1995) Nature 375: 546-549.

Another class of structure modifications include 2'-methylribonucleosides (2'-OMe). Such substitutions are widely described in the state of the art and in particular, with respect to their immunostimulatory properties, in Zhao Q et al. (1999) Bioor Med Chem Lett 9: 3453-8. Zhao et al. describe methods for preparing 2'-OMe modifications in nucleic acids.

The nucleic acid molecules of the invention they can include puric or pyrimidine heterocyclic bases natural or synthetic, as well as modified structures. The bases Pyrrhic or pyrimidine heterocyclics include, but are not limited to a, adenine, guanine, cytosine, thymine, uracil, and inosine. Other Representative heterocyclic bases are shown in the Patent of U.S. No. 3,687,808, published by Merigan, et al. The term purine or pyrimidine or bases are used here to refer to both purines, pyrimidines or natural bases as synthetic.

Other stabilized nucleic acids include: nonionic DNA analogs, such as alkyl- and aryl phosphates (in the that the charged phosphonate oxygen is substituted by an aryl group or alkyl), alkyl phosphodiester and alkyl phosphotriesters, in which the portion of charged oxygen is rented. Nucleic acids containing diol, such as tetraethylene glycol or hexaethylene glycol, in one or both terminal ends, have also shown to be substantially resistant to degradation by nucleases.

According to aspects further from the invention, CpG type nucleic acids are useful for treating to an individual who has or is at risk of developing certain diseases, which include an infectious disease due to a infection with an infectious agent, an allergy or asthma where knows or suspects the allergen or predisposition to asthma, or a cancer in which a carcinogenic antigen has been identified specific. CpG type nucleic acids can also determined without the antigen or allergen for short protection term against infections, cancer, allergy or asthma, and in this case, Repeated doses will allow long-term protection.

The term "treat" is defined as administer to an individual a therapeutically effective amount of a compound (for example, an oligonucleotide of the invention) that It is enough to prevent the onset of, relieve symptoms of, or stop the progression of a disorder or disease that is being treated

An "individual," according to the invention, would mean a human or other vertebrate animal that includes, but does not limit, a dog, cat, horse, cow, pig, sheep, goat, rabbit, guinea pig, rat, mouse, primate no human (eg, monkey), chicken, and fish (fish species, for example, salmon).

An "individual at risk", as used here, is an individual who has a predisposition to have a disease or that has an identified or suspected risk of exposure to an agent or condition associated with the development of an illness. Generally, the risk of developing a particular disease will be greater in the individual at risk than in other individuals that, it is considered, are not at risk.

With reference to an infectious disease, an "individual at risk of developing an infectious disease", as used herein, is an individual who has any identified or suspected risk of exposure to a pathogen that causes the infection. For example, an individual at risk of developing an infection may be an individual who lives or plans to travel to an area where a specific type of infectious agent is found. An individual at risk may be an individual who, due to his or her lifestyle, occupation, or medical procedure, is potentially exposed to bodily fluids or materials that may contain infectious organisms or agents. Alternatively, a risky individual may be an individual who, by his or her lifestyle, occupation, or by a medical procedure, is exposed to infectious organisms or agents. An individual at risk of developing an infection may also be an individual at risk of biological warfare, for example, military personnel or those living in areas at risk of terrorist attack. Individuals at risk of developing an infection also include general populations in which a medical agency recommends vaccination against an infectious organism or agent in
particular.

With reference to allergy or asthma, an individual of risk, as used here, is an individual who has any identified or suspected risk of exposure to a antigen that produces allergy or asthma. For example, an individual with risk of developing allergy or asthma can be an individual who lives or plan to travel to an area where a type of antigen is found or concrete allergen An individual at risk of developing allergy or Asthma can be an individual who, by way of life or employment, It is directly exposed to an antigen or allergen. Individuals at risk of developing allergy or asthma also include general populations in which a medical agency recommends the vaccination with a particular antigen. If the antigen is a allergen and the individual develops seasonal allergic responses against that particular antigen, for example, during pollen station, then the individual may be at risk of develop allergy during the period of exposure to the antigen. An individual at risk of developing an allergy or asthma includes those individuals who have been identified as having a allergy or asthma but who do not have active disease during CpG type nucleic acid treatment, as well as those individuals considered at risk of developing these diseases due to genetic or environmental factors.

With reference to cancer, an "individual with risk of developing cancer "is one that has an increased The chance of having cancer. These individuals include, by For example, individuals who have genetic abnormalities, the its presence has been demonstrated by having a relationship correlative to a greater probability of having cancer, as well as individuals exposed to cancer-causing agents such as tobacco, asbestos, or other chemical toxins, or individuals that have been previously treated against cancer and are in apparent remission. When an individual at risk of developing cancer is treated with a specific antigen for the type of cancer of which the individual is at risk of developing it and a nucleic acid of CpG type, the individual may be able to kill the cells cancerous as they develop. If a tumor begins to form in an individual, the individual will develop a response specific immune against tumor antigen.

In addition to the use of CpG type nucleic acids for prophylactic treatment, the invention also encompasses the use of CpG type nucleic acids for the treatment of an individual You have an infection, an allergy, asthma, or a cancer.

An infectious disease, as used here, it is a disease that arises from the presence of a foreign microorganism or an infectious pathogen in the body. A "individual who has an infectious disease" is an individual  that has been exposed to an infectious pathogen and has levels Acute or chronic pathogen detectable in the body. The Infectious pathogens include viruses, bacteria, fungi, parasites, and other infectious agents. CpG type nucleic acids can be used with an antigen to mount a response mucosal immune or systemic antigen specific that is capable of reducing the level of or eradicating the infectious pathogen.

An individual who has an allergy is a individual who has or is at risk of developing a reaction allergic in response to an allergen. An allergy refers to acquired hypersensitivity to a substance (allergen). The Allergic conditions include, but are not limited to eczema, hives, allergic asthma, allergic rhinitis or coryza, fever hay, conjunctivitis, food allergies, and other conditions atopic

Currently, allergic diseases They are usually treated both symptomatically (see below) and definitely, the latter by injection of small doses of antigen followed by increasing subsequent doses of the antigen. Be believe that this procedure induces allergen tolerance for prevent other allergic reactions However, it can lead several years until these methods can be effective and are associated with the risk of side effects such as anaphylactic shock. The methods of the invention avoid these problems.

Allergies are usually characterized by IgE antibody generation against harmless allergens. The cytokines that are induced by mucosal or systemic administration of CpG type nucleic acids are predominantly of the class called Th1 (examples are IL-2 e IFN-?) And these induce both response humoral immune system (antibodies) as an immune response mobile. The types of antibodies associated with a Th1 response are generally more protective because they have high capacities of neutralization and opsomization. The other immune response majority, which are called Th2 immune response, is associated with the production of IL-4 cytokines, IL-5 and IL-10. The Th2 Answers predominantly involves antibodies and have less effect protective against infection and some Th2 isotypes (for example, IgE) are associated with allergy. In general, it seems that allergic diseases are mediated by immune responses type Th2, while Th1 responses are associated with autoimmune disease. Based on the ability of acids CpG type nuclei to change the immune response in a individual, from a Th2 response (which is associated with production of IgE antibodies and allergy) to a Th1 (which is protective against allergic reactions) can be administered, to an individual, an effective dose of the CpG type nucleic acid for induce an immune response to treat or prevent a allergy.

In this way, CpG type nucleic acids they have significant therapeutic utility in the treatment of Allergic and non-allergic conditions such as asthma. Cytokines Th2, especially IL-4 and IL-5, They are elevated in the airways of asthmatic individuals. These cytokines promote important aspects of the response asthmatic inflammatory, including the exchange of the IgE isotope, chemotaxis and eosinophilic activation, and degranulation of mast cells Th1 cytokines, especially IFN-? And IL-2, can suppress Th2 clone formation and cytokine production Th2.

An individual who has asthma is an individual who have a respiratory system disorder, characterized by inflammation, narrowing of the airways, and increased airway reactivity to agents inhaled Typically, asthma is a chronic disease, characterized by acute reversible episodes of narrowing generalized respiratory tract. Therefore, currently, a individual who has asthma does not require the individual to be symptomatic, that is, you currently have an acute exacerbation of asthma Asthma diagnostic methods are well known in prior art, and these may include measure of reversible airway obstruction in response to pulse with beta-adrenergic agonists, histamines, methacholine, or cold air. Asthma is frequently, although not exclusively, associated with atopic and allergic symptoms.

An individual who has cancer is an individual which has detectable cancer cells. Cancer can be a malignant or non-malignant cancer. Cancers or tumors include, but they are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; neoplasms intraepithelial; lymphomas; Liver cancer; lung cancer (for example, small cell and non-small cell); melanoma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin cancer; Cancer testicular; thyroid cancer; and kidney cancer as well as others carcinomas and sarcomas. In one embodiment, the cancer is leukemia of hairy cell, chronic myelogenous leukemia, cutaneous leukemia T cell, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, carcinoma of prostate, bladder cell carcinoma, or carcinoma of colon.

In some embodiments, the invention may be used to treat cancer and tumors in nonhuman individuals. He cancer is one of the leading causes of death in animals of company (for example, cats and dogs). Normally cancer attacks older animals that, in the case of house pets, They are integrated into the family. Forty-five percent of Dogs older than 10 are susceptible to succumbing to Cancer. The most common treatment options include surgery, Chemotherapy and radiation therapy. Other modalities of treatment that have been used with some success are therapy of laser, cryotherapy, hyperthermia and immunotherapy. The choice of Treatment depends on the type of cancer and the degree of spread. Unless the malignant growth is confined to an area Discreet in the body, it is difficult to remove only the malignant tissue without also affecting normal cells.

Malignant disorders commonly diagnosed in dogs and cats include, but are not limited a, adenoescamoso carcinoma, brain tumor, gland tumor bronchial, bronchiolar adenocarcinoma, Burkitt lymphoma, tumor lung carcinoid, Ewing's sarcoma, fibroma, fibrosarcoma, lymphosarcoma, breast tumors, mastocytoma, melanoma, microglioma, myxochondroma, neuroblastoma, neurosarcoma, oral neoplasia, osteoclastoma, osteoma, osteosarcoma, papilloma, pulmonary sarcoma, retinoblastoma, rhabdomyosarcoma, and Wilms tumor. Other neoplasms in dogs include carcinoma of the adrenal gland, cell tumor baseline, chloroma (granulocytic sarcoma), corneal papilloma, carcinoma squamous cell cornea, cystadenoma, cell carcinoma Genital squamous, hemangioendothelioma, hemangiopericytoma, hemangiosarcoma, histiocytoma, oral papillomatosis, mesothelioma pleural, seminoma, Sertoli cell tumor, stomach tumor, testicular tumor, thymoma, and transmissible venereal tumor. Additional malignancies diagnosed in cats include fibrosarcoma, follicular lymphoma, intestinal lymphosarcoma, and squamous cell carcinoma of the lung. The ferret, the pet more popular, it is known to develop gastric adenocarcinoma, MALT gastric lymphoma, insulinoma, lymphoma, neuroma, cell tumor of pancreatic islet, and sarcoma.

Neoplasms that affect livestock in agriculture include leukemia, hemangiopericytoma, and bovine ocular neoplasia (in cattle); foreskin fibrosarcoma, ulcerative squamous cell carcinoma, foreskin carcinoma, connective tissue neoplasia and mastocytoma (in horses); hepatocellular carcinoma (in pig); lymphoma and pulmonary adenomatosis (in sheep); pulmonary sarcoma, lymphoma, Rous sarcoma, reticulendotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (in avian species); retinoblastoma, liver neoplasia, lymphosarcoma (lymphoblastic lymphoma), plasmacytic leukemia and swim bladder sarcoma (in fish), caseous lymphadenitis (CLA): chronic, infectious, contagious sheep and goat disease caused by Corynebacterium pseudotuberculosis bacteria, and contagious tumor sheep lung caused by jaagsiekte.

CpG type nucleic acids can also be formulated with or administered to an individual in conjunction with an antigen An "antigen," as used herein, is a molecule capable of eliciting an immune response. The antigens include, but are not limited to, cells, extracts cell, proteins, polypeptides, peptides, polysaccharides, conjugates of polysaccharides, peptide and non-peptide mimics of polysaccharides and other molecules, small molecules, lipids, glycolipids, carbohydrates, viruses and viral extracts and organisms multicellular like parasites and allergens. The term antigen widely includes any type of molecule that is recognized as strange for a host's immune system. The antigens include, but are not limited to, cancerous antigens, microbial antigens, and allergens.

The individual is exposed to an antigen. As used herein, with reference to an antigen, the term "exposed" refers to both the active step of contacting the individual with an antigen and the individual's passive exposure to the antigen in vivo . The methods for the active exposure of the individual with an antigen are well known in the state of the art. In general, an antigen is administered directly to the individual by any means such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The antigen can be administered systematically or locally. Methods of administration of the CpG type antigen and nucleic acid are described in more detail below. An individual is passively exposed to an antigen if an antigen is available for exposure to immune cells in the body. An individual may be passively exposed to an antigen, for example, by the entry of a foreign pathogen into the body or by the development of a tumor cell that expresses a foreign antigen on its surface.

The methods by which an individual is passively exposed to an antigen can be particularly time-dependent administration of type nucleic acid CpG For example, in an individual at risk of developing a infectious disease or an allergic or asthmatic response, or a cancer, the individual can be administered with nucleic acid CpG type on a regular basis when said risk is the highest, it is say, during allergy season or after exposure to an agent causing cancer.

In addition, the CpG type nucleic acid can be administered to travelers before their trip to foreign lands, where they are at risk of exposure to infectious agents. So same, the CpG type nucleic acid can be administered to soldiers or civilians at risk of exposure to biological warfare to induce a systemic or mucosal immune response to the antigen when and if the individual is exposed to it.

As used here, the terms "cancerous antigen" and "tumor antigen" are used in a way interchangeable to refer to antigens that are differentially expressed by cancer cells and with it, can be exploited in order to mark cancer cells. Cancerous antigens they are antigens that can potentially stimulate responses seemingly tumor-specific immune Some of these antigens are encoded, although not necessarily expressed, by normal cells These antigens can be characterized as those that are normally silenced (i.e. not expressed) in normal cells, those that are expressed only in certain stages of differentiation, and those that are expressed temporarily as embryonic and fetal antigens. Others cancerous antigens are encoded by mutant cell genes, such as oncogenes (for example, the activated ras oncogene), genes suppressors (e.g., the p53 mutant), and fusion proteins resulting from internal deletions or chromosomal translocations. Also, other cancerous antigens can be encoded by viral genes such as those transported in the RNA and DNA of tumor viruses

A cancerous antigen, as used here, it is a compound, such as a peptide or protein, associated with a cell tumor or cancerous and that is capable of eliciting a response immune when expressed on the surface of a cell cancer in the context of an MHC molecule.

Preferably, the antigen is expressed on the cell surface of the cancer cell. Even more preferred, the antigen is one that is not expressed by normal cells, or at least, not expressed at the same level as in cancer cells. Cancer antigens can be prepared from cancer cells by both preparing crude extracts of cancer cells, for example, as described in Cohen PA et al. (1994) Cancer Research 54: 1055-8; as by partial purification of antigens, by recombinant technology, or by de novo synthesis of known antigens. Cancerous antigens can be used in the form of immunogenic portions of a particular antigen or, in some cases, a whole cell or a tumor mass can be used as the antigen. Cancerous antigens include, but are not limited to, antigens that are recombinantly expressed, an immunogenic portion of a cancer cell or tumor, or a complete tumor or cancer.

A microbial antigen, as used here, it is an antigen of a microorganism and includes, but is not limited to, antigens associated with viruses, bacteria, fungi, and parasites Such antigens include the intact microorganism as well. as isolated and natural fragments or derivatives thereof and also synthetic compounds that are identical or similar to antigens of the natural microorganism, and induce an immune response specific for that microorganism. A compound is similar to a antigen of a natural microorganism if it induces a immune response (humoral and / or cellular) of an antigen from a natural microorganism These antigens are used in a way routine in the state of the art and are well known to experts in the art.

Examples of infectious viruses include, but are not limited to: Adenoviridae (most adenoviruses); Arena viridae (hemorrhagic fever virus); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Bungaviridae (for example, Hantaan virus, bunga virus, flebovirus and Nairo virus); Calciviridae (for example, strains that cause gastroenteritis); Coronaviridae (for example, coronavirus); Filoviridae (for example, Ebola virus); Flaviridae (for example, dengue virus, encephalitis virus, yellow fever virus); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus); Iridoviridae (for example, African pig fever virus); Orthomyxoviridae (for example, influenza virus); Paramyxoviridae (for example, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Picornaviridae (for example, polio virus, Hepatitis A virus, enterovirus, human Coxsackie virus, rhinovirus, ecovirus); Poxviridae (variola virus, vaccinia virus, pox virus); Reoviridae (for example, reovirus, orbivirus and rotavirus); Retroviridae (for example, human immunodeficiency virus, such as HIV-1 (also called HTLV-III, LAV or HTLV-III / LAV, or HIV-III, and other isolates, such as HIV-LP)); Rhabdoviridae (for example, vesicular stomatitis virus, rabies virus); Togaviridae (for example, equine encephalitis virus, rubella virus); and unclassified viruses (for example, the etiologic agent of spongiform encephalopathies, the hepatitis delta agent (thought to be a defective satellite of the Hepatitis B virus), non-A, non-B hepatitis agents (class1 = internally transmitted; class2 = parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria include, but are not limited to: Acinetobacter Spp., Actinomyces israelli, Bacillus anthracis, Bacteroides spp., Bordetella pertussis, Borrelia burgdorfeli, Brucell melitensis, Campylobacter spp. pathogenic, clostridium difficile, Clostridium perfringens, Clostridium tetan, Corynebacterium diphtheriae , other Corynebacterium spp. Enterobacter aerogenes, Enterococcus spp., Erysipelothrix rhusiophatiae, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pilori, Klebsiella pneumoniae, Legionella pneumophilia, Leptospira spp., Listeria monogenes, Tuberculosis. M. avium, M. gordonae, M. intracellulare, and M. kansasii), Neisseria gonorrheoae, Neisseria meningitidis, Nocardia asteroides, Nocardia brasiliensis, Pasturella multocida, Peptostreptococcus spp., Proteus spp., Pseudomonas aeruginosa, other Pseudomonas spp., Rickettsia , Salmonella spp., Serratia spp., Shigella spp., Staphylococcus aureus, Streptobacillus moniliformis, Streptococcus (ssp. Aeróbica), Streptococcus (viridans group), Streptococcus agalactiae (group B Streptococcus), Streptococcus bovis, Streptococcus faecalis, Streptococcus pneumoniae, Streptococcus pyrogenes (Streptococcus Group A), Treponema pallidum, Treponema pertenue, Vibrio Cholerae, other spp. Vibrio , and Yersinis spp.

Examples of infectious fungi include, but are not limited to: Aspergillus spp., Blastomices dermatitidis, Candida albicans, other Candida spp., Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, and Rhizopus spp.

Other infectious organisms include, but are not limited to: Babesia divergens, Babesia microti, Chlamydia trachomatis, Giardia spp., Leishmania braziliensis, Leishmania donovani, Leishmania major, Leishmania tropica, Plasmodium spp. (For example, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium knowlesi, Plasmodium knowlesi, malariae, Plasmodium ovale, and Plasmodium vivax ), Toxoplasma gondii, Trichinella spiralis, Trypanosoma cruzi, Trypanosoma gambiense, and Trypanosoma Rhodesiense.

Other medically relevant microorganisms have been widely described in the literature, for example, see CGA Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983.

Although many of the microorganisms above described relate to human diseases, the invention It is also useful for treating non-human vertebrates. Vertebrates nonhumans are also able to develop infections that they can be prevented or treated with nucleic acids of type CpG described here. For example, in addition to the treatment of Human infectious diseases, the methods of the invention are Useful for treating animal infections.

In Bennett, K., Compendium of Veterinary Products , 3rd ed., North American Compendiums, Inc., 1995, many vaccines for the treatment of non-human vertebrates are described.

As discussed above, the antigens include infectious microbes such as viruses, bacteria, parasites, fungi, and fragments thereof, derived from natural sources or synthetic

Infectious human and vertebrate viruses non-human, include retroviruses, RNA viruses and DNA viruses. The group of retrovirus includes the subgroup of type B retrovirus, retrovirus type C and type D retrovirus. An example of type retrovirus B is the mouse mammary tumor virus (MMTV). The retroviruses of type C include subgroups of group A of type C (which includes the Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avian myeloblastosis virus (AMV)) and group B type C (which includes feline leukemia virus (FeLV), leukemia virus gibbon ape (GALV), spleen necrosis virus (SNV), reticuloendotheliosis (RV) and ape sarcoma virus (SSV). The Type D retroviruses include the Mason-Pfizer virus of monkeys (MPMV) and type 1 retrovirus of apes (SRV-1). Complex retroviruses include the subgroups of lentiviruses, T-cell leukemia viruses and viruses sparkling

Lentiviruses include HIV-1, HIV-2, SIV, Visna virus, feline immunodeficiency (IVF), and equine infectious anemia virus (EIAV). T-cell leukemia viruses include HTLV-1, HTLV-II, leukemia virus of apes T cell (STLV), and bovine leukemia virus (BLV). The Sparkling viruses include human foamy virus (HFV), virus Sparkling Apes (SFV) and Sparkling Bovine Viruses (BFV).

Examples of other RNA viruses that are antigens in vertebrate animals include, but are not limited to, members of the family Reoviridae, including the genus Orthoreovirus (multiple serotypes of retroviruses, both from mammals and from birds), the genus Orbivirus (Bluetongue virus, Eugenangee virus Kemerovo, African horse disease virus, and virus Colorado Tick Fever), the genus Rotavirus (human rotavirus, virus Nebraska of baby diarrhea, ape rotavirus, rotavirus sheep or cattle, bird rotavirus); the Picornaviridae family, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, human enterocytopathic orphan virus (ECHO), hepatitis A, ape enterovirus, encephalomyelitis virus murides (ME) Poliovirus muris, bovine enterovirus, enterovirus swine, the genus Cardiovirus (encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus (human rhinoviruses that include at least 113 subtypes; other rhinoviruses), the genus Apthovirus (foot and mouth disease (FMDV); Calciviridae family, including pig vesicular rash virus, lion virus San Miguel sailor, feline picornavirus and Norwalk virus; the Togaviridae family, including the genus Alphavirus (virus West equine encephalitis, Semliki forest virus, virus Sindbi, Chikungunya virus, O'Nyong-Nyong virus, Ross River virus, equine encephalitis virus from Venezuela, West equine encephalitis virus), the genus Flavivirus (mosquito terminal yellow fever virus, Dengue virus, Japanese encephalitis virus, St encephalitis virus, Louis, Murray Valley encephalitis virus, west virus Nile, Kunjin virus, Central European terminal Tick virus, Tick virus far west terminal, Kyasanur forest virus, Louping virus III, Powassan virus, Omsk hemorrhagic fever virus), the Genus Robivirus (Rubella virus), the genus Pestivirus (virus mucosal disease, cholera virus Hog, disease virus Border); the Bunyaviridae family, including the genus Bunyvirus (Bunyamwera virus and related, encephalitis group virus of California), the genus Phlebovirus (Sicilian fever virus sandfky, Rift Valley fever virus), the genus Nairovirus (vieru from the Crimean-Congo hemorrhagic fever, Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi virus and related); the Orthomyxoviridae family, including the genus of Influenza virus (Influenza type A virus, many human subtypes); swine influenza virus, and virus avian and equine influenza; type B influenza (many subtypes humans), and influenza type C (possible separate genera); the paramyxoviridae family, including the genus Paramyxovirus (virus Parainfluenza type 1, Sendai virus, Hemadsorption virus, virus Parainfluenza types 2 to 5, Newcastle Disease Virus, Mumps virus), the genus Morbillivirus (measles virus, Subcutaneous sclerosing panencephalitis virus, distemper, Rinderpest virus), the genus Pneumovirus (virus respiratory syncytial (RSV), respiratory syncytial virus bovine and Pneumonia virus); the Rhabdoviridae family, including the genus Vesiculovirus (VSV), Chandipura virus, virus Flanders-Hart Park), the genus Lyssavirus (virus Rabies), Rhabdovirus fish, and two probable Rhabdovirus (viruses Marburg and Ebola virus); the Arenaviridae family, including viruses of lymphocytic choriomeningitis (LCM), the Tacaribe virus complex, and Lassa virus; the Coronoaviridae family, including Virus Infectious bronchitis (IBV), Hepatitis virus, enteric virus crown human, and feline infectious peritonitis (feline coronavirus).

Illustrative DNA viruses that are antigens in vertebrate animals include, but are not limited to, the Poxviridae family, including the genus Orthopoxvirus (Variola major, Variola minor, pox Vaccinia monkey, cow pox, bux pox, rabbit pox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (poultry pox, other bird poxvirus), the genus Capripoxvirus (sheep pox, goat pox), the genus Suipoxvirus (pig pox), the genus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox, bovine postomatic stomatitis virus); the Iridoviridae family (African pig fever virus, frog virus 2 and 3, fish lymphocystis virus); Herpesviridae family, including alpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster, equine abortion virus, equine herpes virus 2 and 3, pseudorabia virus, bovine infectious keratoconjunctivitis virus, bovine infectious rhinotracheitis virus , feline rhinotracheitis virus, infectious laryngotracheitis virus) Beta-herpesviruses (human cytomegalovirus and pig and monkey cytomegalovirus); gamma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus, Herpes saimiri, Herpesvirus ateles, Herpesvirus sylvilagus, herpes guinea pig virus, Lucke tumor virus); the Adenoviridae family, including the genus Mastadenovirus (human subgroups A, B, C, D, E and non-grouped ape adenoviruses (at least 23 serotypes), canine infectious hepatitis, and adenovirus of cattle, pigs, sheep, frogs and many other species, the genus Aviadenovirus (avian adenovirus); and non-cultivable adenovirus; the Papoviridae family, including the genus Papillomavirus (human papillomavirus, bovine papillomavirus, Shope rabbit papillomavirus, and several other pathogenic papillomavirus species), the genus Polyomavirus (polyomavirus, ape vacuolating agent (SV-40), rabbit vacuolating agent (RKV), K virus, BK virus, JC virus, and other primate virus polyoma such as lymphotropic virus papilloma); Parvoviridae family including the genus of Adeno-associates virus, the genus Parvovirus (feline panleucopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc.) Finally, DNA viruses they can include cirus that do not fit in the families above, such as Kuru and Creutzfeldt-Jacob disease viruses and chronic infectious neuropathic agents (viruses
CHINA).

Each of the preceding lists are illustrative, and are not intended to be limiting.

In addition to the use of type nucleic acids CpG to induce an antigen specific immune response in humans, the methods of preferred embodiments are particularly suitable for the treatment of birds such as chickens, chickens, turkeys, ducks, geese, quail, and pheasant. The Birds are the main target of many types of infections.

The brooding birds are exposed to pathogenic microorganisms after birth. Although initially,  these birds are protected against pathogens by antibodies derived from the mother, this protection is only temporary, and the own immature immune system of birds should start to Protect the bird against pathogens. Normally, it is desirable. prevent infection in young birds when they are older susceptible. It is also desirable to prevent infection in birds older, especially when birds stay in small spaces, causing the rapid spread of the disease. By it is desirable to administer the CpG type nucleic acid of the invention to birds that develop an immune response antigen specific when the antigen is present.

An example of a common viral infection in chickens is the chicken infectious anemia virus (CIAV). CIAV was first isolated in Japan in 1979 during an investigation of interruption of Marek's disease vaccination. Yuasa et al. (1979) - Avian Dis 23: 366-385. Since that time, CIAV had been detected in commercial poultry in all poultry producing countries (van Bulow et al., In Diseases of Poultry , 9th edition, Iowa State University Press, 1991, pp. 690-699) .

CIAV infection resulted in a disease clinic characterized by anemia, hemorrhage and immunosuppression, in susceptible young chickens. They are also characteristics of the CIAV infection, thymus and bone marrow atrophy and consistent lesions in chickens infected with CIAV. Depletion of lymphocytes in the thymus, and occasionally in the bursa of Fabricius, results in immunosuppression and increased susceptibility to infections  secondary viral, bacterial, or fungal that with it, complicate the course of the disease. Immunosuppression can cause aggravated disease after infection with one or more viruses Marek's disease (MDV), with bursal disease virus infectious, with reticuloendotheliosis virus, with adenovirus, or reovirus It has been shown that the pathogenesis of MDV is increased by CIAV (DeBoer et al., 1989, p. 28, In: Proceedings of the 38th Western Poultry Diseases Conference, Tempe, Ariz.).

In addition, CIAV has been shown to aggravate the signs of infectious bursal disease. Rosenberger et al. (1989) Avian Dis 33: 707-713. With age, chickens develop an experimentally induced resistance to disease due to CAA. This is essentially complete at the age of 12 weeks, but older birds are still susceptible to infection (Yuasa, N. et al., 1979 above ; Yuasa, N. et al., Avian Dis 24, 202-209, 1980). However, if chickens are infected twice with CAA and an immunosuppressive agent (IBDV, MDV, etc.), resistance with age against the disease is delayed (Yuasa, N. et al., 1979 and 1980 above ; Bulow von V. et al. J Veterinary Medicine 33: 93-116,1986). The characteristics of CIAV that could enhance disease transmission include high resistance to environmental inactivation and some common disinfectants. The economic impact of CIAV infection in the poultry industry is clear from the fact that 10% to 30% of infected birds die in disease outbreaks.

Vaccination of birds, like other vertebrate animals, can be performed at any age. Normally, vaccinations are performed after 12 weeks of age for a live microorganism and between 14-18 weeks for an inactivated microorganism or other type of vaccine. For in ovo vaccination, vaccination can be performed in the last quarter of embryonic development. The vaccine can be administered subcutaneously, by spray, orally, intraocularly, intratracheally, nasally, or by any other mucosal administration method described herein. Therefore, the CpG type nucleic acids of the invention can be administered to birds and other non-human vertebrates using routine vaccination programs and the antigen can be administered after an appropriate period of time as described herein.

Livestock and livestock properties too They are susceptible to infection. The diseases that affect these animals can produce severe economic losses, especially Against cattle The methods of the invention can be used. to protect against infection in cattle, such as cows, horses, Pigs, sheep, and goats.

Cows can be infected by viruses bovines The bovine viral diarrhea virus (BVDV) is a virus small envelope positive RNA chain and is classified together with the hog cholera virus (OCT) and the disease virus sheep (BDV), in the genus Pestivirus. Although previously they classified the Pestiviruses in the Togaviridae family, Some studies have suggested reclassification within the Flaviviridae family together with the flavivirus and virus group of hepatitis C (HCV) (Francki, et al., 1991).

BVDV, which is an important pathogen in the cattle, can be distinguished, based on crop analysis cellular, in cytopathogenic (CP) and non-cytopathogenic biotypes (NCP) The NCP biotype is more widespread, although both biotypes are They can find in cattle. If a pregnant cow becomes infected with an NCP strain, the cow can give birth to an infected baby persistently and specifically immunotolerant that will extend the virus during its lifetime. Infected cattle persistent can succumb to mucosal diseases, and both Biotypes can then be isolated from the animal. The Clinical minifestations may include abortion, teratogenesis, and respiratory problems, mucosal disease and mild diarrhea. Further, severe thrombocytopenia has been described, associated with epidemics of the pack, which can result in the death of the animal, and the strains associated with this disease appear to be more virulent than the classic BVDVs.

Equine herpes viruses (EHV) comprise a group of antigenically different biological agents that cause a variety of infections in horses, from a subclinical range to a fatal disease. These include equine herpesviruses (1 EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated with abortion epidemics, respiratory tract diseases, and central nervous system disorders. The main infection of the upper respiratory tract of young horses results in a febrile illness that lasts 8 to 10 days. Immunologically experienced mares can be re-infected via the respiratory tract without apparent disease, so abortion occurs without warning. The neurological syndrome is associated with respiratory disease or abortion and can affect animals of any sex and age, causing lack of co-ordination, weakness and subsequent paralysis. Telford EAR et al. (1992) Virology 189: 304-316. Other EHVs include EHV-2, or equine cytomegalovirus, EHV-3, equine coital rash virus, and EHV-4, previously classified as EHV-1 subtype 2.

Sheep and goats can be infected by a variety of dangerous microorganisms that include visna-maedi.

Primates such as monkeys, apes and macaques can be infected by simian immunodeficiency virus (SIV). Inactivated ape vaccines of cellular immunodeficiency virus and total free cells have been demonstrated to allow protection in macaques. Stott et al. (1990) Lancet 36: 1538-1541; Desrosiers et al. (1989) Proc Natl Acad Sci USA 86: 6353-6357; Murphey-Corb et al. (1989) Science 246: 1293-1297; and Carlson et al. (1990) AIDS Res Human Retroviruses 6: 1239-1246. A recombinant HIV gp120 vaccine has been shown to allow protection in chimpanzees. Berman et al. (1990) Nature 345: 622-625.

Cats, both domestic and wild, are susceptible to infection with a variety of microorganisms. By example, Feline infectious peritonitis is a disease that occurs in both domestic and wild cats, such as lions, Leopards, cheetahs, and jaguars. When you want to prevent infection with this or other types of pathogenic organisms in cats, the methods of the invention can be used to vaccinate Cats to protect them against infection.

Domestic cats become infected with several retroviruses, which include, but are not limited to, viruses feline leukemia (FeLV), feline sarcoma virus (FeSV), Endogenous Concornavirus (RD-114), and virus Feline Syncytium Formation (FeSFV). Of these, FeLV is the most significant pathogen, because it causes various symptoms, which include lymphoretic and myeloid neoplasms, anemias, mid immune disorders, and an immunodeficiency syndrome that It is similar to human acquired immunodeficiency syndrome (AIDS). Recently, a particular mutant of FeLV defective in the replication, called FeLV-AIDS, has been associated more particularly with immunosuppressive properties.

The discovery of the feline T-lymphocytic lentivirus (also called feline immunodeficiency virus, IVF) was first demonstrated by Pedersen et al. (1987) Science 235: 790-793. Characteristics of IVF have been demonstrated in Yamamoto et al. (1988) Leukemia 2 (12 Supp): 204S-215S; Yamamoto et al. (1988) Am J Vet Res 49: 1246-1258; and Ackley et al. (1990) J Virol 64: 5652-5655. Cloning and IVF sequence analysis has been demonstrated in Olmsted et al. (1989) Proc Natl Acad Sci USA 86: 2448-2452 and 86: 4355-4360.

Feline infectious peritonitis (FIP) is a sporadic disease that occurs unpredictably in Felids Domestic and wild. While FIP is primarily a domestic cat disease, has been diagnosed in lions, mountain lions, leopards, cheetahs, and in the jaguar. The cats smaller savages that have been affected by FIP include the Lynx and caracal, the sand cat, and the pallas cat. In cats Domestic, the disease occurs predominantly in animals young, although cats of any age are susceptible. A Peak incidence occurs between 6 and 12 months of age. Be warns a decrease in incidence from 5 to 13 years of age, followed by an increase in the incidence in cats from 14 to 15 years old.

Viral, bacterial and parasitic in finfish, shellfish or other life forms aquatic, pose a serious problem for the industry fish farm. Due to the high density of animals in the fish farms or fish farms, diseases Infectious can eradicate a large proportion of the reserve, for example, finfish, shellfish, or other life forms aquatic Disease prevention is the most desired remedy for these threats in fish, more than intervention once That the disease is in progress. The vaccination of fish is the only preventive method, which can offer long protection term through immunity. Acid based vaccines Nuclei are described in US Patent No. 5,780,448 issued by Davis.

Fish species include, but not They are limited to finfish, shellfish, and other aquatic animals. Finned fish include all vertebrate fish, which can be fish with bone or cartilaginous, such as Salmonids, carp, catfish, yellowtail, sea bream, and sea bass. The Salmoids are a family of finfish that include trout (including rainbow trout), salmon, and Arctic char.

Examples of seafood include but are not limited to to, clams, lobster, shrimp, crab, and oysters. Other animals Fish farms include, but are not limited to, eels, squid and octopus.

The polypeptides of the viral pathogens of the Fish farms include, but are not limited to, glycoprotein (G) or Nucleoprotein (N) viral haemorrhagic septicemia virus (VHSV); G or N proteins of the hematopytic necrosis virus infectious (IHNV); structural proteins VP1, VP2, VP3 or N of the infectious pancreatic necrosis virus (IPNV); G protein of spring carp viremia (SVC), and a protein associated with membrane, tegumin or capsid protein or glycoprotein of Channel catfish virus (CCV).

Typical parasites that infect horses are Gasterophilus spp .; Eimeria leuckarti; Giardia spp .; Tritrichomonas equi; Babesia spp. (RBC's); Theileria equi; Trypanosoma spp .; Klossiella equi; and Sarcocystis spp.

Typical parasites that infect pigs include Eimeria bebliecki; Eimeria scabra; Isospora suis; Giardia spp .; Balantidium coli; Entamoeba histolytica; Toxoplasma gondii and Sarcocystis spp .; and Trichinella spiralis.

The main parasites of the young of cows and cows include Eimeria spp .; Cryptosporidium spp .; Giardia spp .; Toxoplasma gondii; Babesia bovis (RBC); Babesia bigemina (RBC); Trypanosoma spp. (plasma); Theileria spp. (RBC); Theileria parva (lymphocytes); Tritrichomonas foetus; and Sarcocystis spp.

The main parasites of birds of prey include Trichomonas gallinae; Coccidia ( Eimeria spp.); Plasmodium relictum; Leucocytozoon danilewskyi (owls); Haemoproteus spp .; Trypanosoma spp .; Histomonas; Cryptosporidium meleagridis; Cryptosporidium baileyi; Giardia; Eimeria; Toxoplasma

Typical parasites that infect sheep and goats include Eimeria spp .; Cryptosporidium spp .; Giardia spp .; Toxoplasma gondii; Babesia spp. (RBC); Trypanosoma spp. (plasma); Theileria spp. (RBC); and Sarcocystis spp.

Poultry parasitic infections include coccidiosis caused by Eimeria acervulina; E. necatrix; E. tenella; Isospora spp. and Eimeria truncata; histomoniasis; caused by Histomonas meleagridis and Histomonas gallinarum; trichomoniasis caused by Trichomonas gallinae; and hexamithiasis caused by Hexamita meleagridis. Poultry can also be infected by Emeria maxima; Emeria meleagridis; Eimeria adenoeides; Eimeria meleagrimitis; Cryptosporidium; Eimeria brunetti; Adenoeides emerge; Leucocytozoon spp .; Plasmodium spp .; Hemoproteus meleagridis; Toxoplasma gondii; and Sarcocystis.

The methods of the invention can also be applied for the treatment and / or prevention of parasitic infections in dogs, cats, birds, fish and ferrets. Typical parasites of birds include Trichomonas gallinae; Eimeria spp .; Isospora spp .; Giardia; Cryptosporidium; Sarcocystis spp .; Toxoplasma gondii; Haemoproteus / Parahaemoproteus; Plasmodium spp .; Leucocytozoon / Akiba; Atoxoplasma; and Trypanosoma spp. Typical parasites that infect dogs include Trichinella spiralis; Isopora spp .; Sarcocystis spp .; Cryptosporidium spp .; Hammondia spp .; Giardia duodenalis (canis); Balantidium coli; Entamoeba histolytica; Hepatozoon canis; Toxoplasma gondii; Trypanosoma cruzi; Babesia canis; Leishmania amastigotes; and Neospora caninum.

Typical parasites that infect feline species include Isospora spp .; Toxoplasma gondii; Sarcocystis spp .; Hammondia hammondi; Besnoitia spp .; Giardia spp .; Entamoeba histolytica; Hepatozoon canis; Cytauxzoon spp .; Cytauxzoon spp .; and Cytauxzoon spp. (red cells; RE cells).

Typical parasites that infect fish include Hexamita spp .; Eimeria spp .; Cryptobia spp .; Nosema spp .; Myxosoma spp .; Chilodonella spp .; Trichodina spp .; Plistophora spp .; Myxosoma Henneguya; Costia spp .; Ichthyophithirius spp .; and Oodinium spp.

Typical parasites of wild mammals include Giardia spp. (carnivores; herbivores); Isospora spp. (carnivores); Eimeria spp. (carnivores; herbivores); Theileria spp. (herbivores); Babesia spp. (carnivores; herbivores); Trypanosoma spp. (carnivores; herbivores); Schistosoma spp. (herbivores); Fasciola hepatica (herbivores); Fascioloides magna (herbivores); Gigantic fasciola (herbivores); and Trichinella spiralis (carnivores: herbivores).

Parasitic infections in zoos can also pose serious problems. Typical parasites of the family bovidae (blesbok, antelope, buffalo, eland, impala, klipsringer, kudu, gazelle) include Eimeria spp. Typical parasites in the pinnipedae family (seal, sea lion) include Eimeria Phocae . Typical parasites of the family camelidae (camels, llamas) include Eimeria spp. Typical parasites of the giraffldae family (giraffes) include Eimeria spp. Typical parasites in the elephantidae family (African and Asian) include Fasciola spp. Typical parasites of lower primates (chimpanzees, orangutans, apes, baboons, macaques, monkeys) include Giardia spp .; Balantidium coli; Entamoeba histolytica; Sarcocystis spp .; Toxoplasma gondii; Plasmodim spp. (RBC); Babesia spp. (RBC); Trypanosoma spp. (plasma); and Leishmania spp. (macrophages).

Bacterial pathogen polypeptides include, but are not limited to, an iron-regulated outer membrane protein (IROMP), an outer membrane protein (OMP), and an Aeromonis salmonicide -A protein that causes furunculosis, p57 protein of Renibacterium salmoninarum that causes bacterial kidney disease (BKD), surface-associated major antigen (msa), a surface-expressed cytotoxin (mpr), a surface-expressed hemolysin (ish) and a flagellar Yersiniosis antigen; an extracellular protein (ECP) an iron-regulated outer membrane protein (IROMP), and a structural Pasteurellosis protein; an OMP and a flagellar protein of Fibrosis anguillarum and V. ordalii; a flagellar protein, an OMP protein, aroA, and purA from Edwardsiellosis ictaluri and E. tard ; and a surface antigen from Ichthyophthirius; and a structural and regulatory protein of Cytophaga columnari; and a structural and regulatory protein from Rickettsia.

Polypeptides of a parasitic pathogen include, but are not limited to the surface antigens of Ichthyophthirius.

CpG type nucleic acids can also be formulated with, or administered to an individual in conjunction with an allergen. An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible individual. The list of allergens is huge and can include pollens, insect poisons, scales, animal hair and feathers, dust, fungal spores and medications (for example, penicillin). Examples of animal and plant allergens include, but are not limited to specific proteins of the following genera: Agropyron (eg, Agropyron repens ); Agrostis (for example, Agrostis alba ); Alder; Alnus ( Alnus gultinoasd ); Alternaria ( Alternaria alternata ); Ambrosia ( Ambrosia artemiisfolia ); Anthoxanthum (for example, Anthoxanthum odoratum ); Apis (for example, Apis multiflorum ); Arrhenatherum (for example, Arrhenatherum elatius ); Artemisia ( Artemisia vulgaris ); Oatmeal (for example, Avena sativa ); Betula ( Betula verrucosa ); Blattella (for example, Blattella germanica ); Bromus (for example, Bromus inermis ); Canine ( Canis familiar is ); Chamaecyparis (for example, Chamaecyparis obtusa ); Cryptomeria ( Cryptomeria japonica ); Cupressus (for example, Cupressus sempervirens, Cupressus arizonica, and Cupressus macrocarpa ); Dactylis (for example, Dactylis glomerata ); Dermatophagoides (for example, Dermatophagoides farinae ); Felis ( Felis domesticus ); Festuca (for example, Festuca elatior ); Holcus (for example, Holcus lanatus ); Juniperus (for example, Juniperus sabinoides, Juniperus virginiana, Juniperus communis, and Juniperus ashei ); Lolium (for example, Lolium perenne or Lolium multiflorum ); Olea ( Olea Europa ); Parietaria (for example, Parietaria officinalis or Parietaria judaica ); Paspalum (for example, Paspalum notatum ); Periplanet (for example, American Periplanet ); Phalaris (for example, Phalaris arundinacea ); Phleum (for example, Phleum pratense ); Plantago (for example, Plantago lanceolate ); Poa (for example, Poa pratensis ox Poa compresssa ); Quercus ( Quercus alba ); Secale (for example, Secale cereale ); Sorghum (for example, Sorghum halepensis ); Thuya (for example, Thuya orientalis ); and Triticum (for example, Triticum aestivum ).

       \ global \ parskip0.850000 \ baselineskip
    

The antigen may be an antigen that is encoded by a nucleic acid vector or may not be encoded by a nucleic acid vector. In the first case, the nucleic acid vector is administered to the individual and the antigen is expressed in vivo . In the second case, the antigen can be administered directly to the individual. An antigen not encoded in a nucleic acid vector, as used herein, refers to any type of antigen that is not a nucleic acid. For example, in some aspects of the invention, the antigen not encoded in a nucleic acid vector is a polypeptide. Small modifications of the main amino acid sequences of polypeptide antigens can also result in a polypeptide that has substantially equivalent antigenic activity compared to the unmodified homologous polypeptide. Such modifications may be deliberate, such as site-directed mutagenesis, or they may be spontaneous. All polypeptides produced by these modifications are included here while there is still antigenicity. The polypeptide can be, for example, a viral polypeptide.

The term "substantially purified", as used herein, refers to the state of a compound in particularly because it is substantially free of others proteins, lipids, carbohydrates or other materials with which It is naturally associated. The term substantially purified, as used herein, applied to a polypeptide, therefore, it refers to a polypeptide that is substantially free of others proteins, lipids, carbohydrates or other materials with which It is naturally associated. A person skilled in the art can purify viral or bacterial polypeptides using techniques Classic for protein purification. Polypeptide substantially pure will normally produce a main band in a non-reducing polyacrylamide gel. In the case of polypeptides partially glycosylated or those that have several codons of Start, there will be several bands in a polyacrylamide gel not reducer, but these will form a distinctive pattern for that polypeptide. The purity of the viral or bacterial polypeptide also can be determined by amino acid amino acid sequence analysis terminal. Other types of antigens not encoded by a vector of nucleic acid such as polysaccharides, small molecules, mimics, etc., are also described above and are included in the invention.

The invention also uses polynucleotides encoding antigenic polypeptides. It is envisioned that the antigen can be delivered to an individual in a nucleic acid molecule encoding the antigen, such that the antigen must be expressed in vivo . Said antigens supplied to the individual in a nucleic acid vector refer to antigens encoded by a nucleic acid vector. The nucleic acid encoding the antigen is operably linked to a gene expression sequence that directs the expression of the antigenic nucleic acid within the eukaryotic cell. The gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or a promoter-enhancer combination, which facilitates the efficient transcription and translation of the antigenic nucleic acid to which it is operatively linked. The gene expression sequence may, for example, be a mammalian or virus promoter, as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter, and other constitutive promoters. Examples of viral promoters that function constitutively in eukaryotic cells include, for example, cytomegalovirus (CMV), ape virus promoters, (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma, long terminal repeats (LTS) of Moloney leukemia virus and other retroviruses, and the thymidite kinase promoter of herpes simplex virus. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

In general, the gene expression sequence should include, as necessary, 5 'non-transcribed and 5' sequences untranslated involved in the initiation of transcription and translation, respectively, as TATA boxes, hood sequences, CAAT sequence, and the like. Especially, the 5 'sequences do not transcribed will include a promoter region that includes a sequence promoter for nucleic acid transcriptional control Operationally bound antigen. As desired, the sequences of gene expression optionally include enhancer sequences or upstream activating sequences.

The antigenic nucleic acid is operatively linked to the gene expression sequence. As used here, the antigenic nucleic acid sequence and the sequence of gene expression is said to be operably linked when are covalently linked in a way that takes place transcription and / or translation of the sequence encoding the antigen under the influence or control of the sequence of gene expression. It is said that there are two DNA sequences that are operably linked if the induction of a promoter in the 5 'gene expression sequence results in the transcription of the antigenic sequence and if the nature of the union between the two DNA sequences not (1) causes the introduction of a mutation of frame change, (2) interferes with the ability of the region promoter to direct the transcription of the antigenic sequence, or (3) interferes with the ability of the corresponding transcript of RNA to be translated into a protein. Therefore, a sequence of gene expression can be operably linked to a sequence of antigenic nucleic acid if the gene expression sequence were capable of transcribing that acid sequence antigenic nucleic such that the resulting transcript is translated to the desired protein or polypeptide.

The antigenic nucleic acid of the invention it can be supplied to the immune system alone or associated with a vector. In its broadest sense, a vector is any vehicle capable of facilitating the transfer of antigenic nucleic acid to immune system cells, so that the antigen can be expressed and presented on the surface of the immune cell. He vector, usually transports nucleic acid to cells immune with reduced degradation related to the extent of degradation that could result from the absence of the vector. He vector optionally includes the gene expression sequence described above to enhance nucleic acid expression antigen in immune cells. In general, useful vectors in the invention include, but are not limited to, plasmids, phagomids, viruses, other vehicles derived from viral sources or bacteria that have been manipulated by insertion or incorporation of antigenic nucleic acid sequences.

Viral vectors are a preferred type of vector and includes, but is not limited to, nucleic acid sequences of the following viruses: retroviruses, such as leukemia virus Moloney of Murids, Harvey Murid Sarcoma Virus, Virus of mammary murine tumor, and Rous sarcoma virus; adenovirus; adeno-associated viruses; type virus SV-40; polyoma virus; virus Epstein-Barr; papillomavirus; herpes virus; virus vaccinia; polio virus; and RNA viruses such as retroviruses. One can, easily use other vectors not named here but known in the state of the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced by the gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of viral genomic RNA to DNA with subsequent proviral integration into the host's cellular DNA. Retroviruses have been approved for human gene therapy trials. The most useful are those retroviruses that are deficient in replication (ie, capable of directing the synthesis of desired proteins, but unable to make an infectious particle). Such genetically altered retroviral expression vectors have general utility for high efficiency gene transduction in vivo . Classical protocols for the production of replication-deficient retroviruses (including the steps of incorporating exogenous genetic material into a plasmid, transfection with the plasmid of a packaging cell line, production by recombinant retrovirus packaging cell line, collection of the medium Tissue culture of viral particles, and infection of target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, WH Freeman Co., New York (1990) and Murry, EJ, Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991).

A preferred virus for certain applications is adeno-associated virus (AAV), a double virus DNA chain The adeno-associated virus can be manipulated to be replication deficient and is able to infect a wide range of cell types and species. Further, It has advantages such as stability in hot solutions and lipid; high transduction frequencies in various cells lineages, including hematopoietic cells; and lack of inhibition superinfection, thereby allowing multiple series of transduction It has been shown that viruses adeno-associates can be integrated into DNA human cell in a specific way; minimizing, of that way, the possibility of insertional mutagenesis and the variability of the expression of the inserted gene, characteristic of retroviral infection In addition, virus infections wild adeno-associates have been followed in tissue culture for more than 100 passes in the absence of pressure selective, assuming the genomic integration of the virus adeno-associated is a relatively event stable. Adeno-associated viruses can also function in an extrachromosomal manner.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the state of the art and are well known in the state of the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual . Second Edition, Cold Spring Harbor Laboratory Press, 1989. In recent years, plasmid vectors have been particularly advantageous in delivering genes, in vivo, to cells due to their inability to replicate and integrate into the host genome. However, these plasmids, which have a promoter compatible with the host cell, can express a peptide of a gene operably encoded within the plasmid. Some of the commonly used plasmids include pBR322, pUC18, pUC19, pRc / CMV, pRc / RSV, pcDNA3.1, pcDNA4, pZeoSV2, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. In addition, plasmids can be designed using restriction enzymes and ligation reactions to remove and add specific DNA fragments.

Recently it has been discovered that plasmids that carry genes can be supplied to the immune system using bacteria. Modified forms of bacteria such as Salmonella can be transfected with the plasmid and used as delivery vehicles. Bacterial delivery vehicles can be administered to a host individual orally or by other means of administration. The bacterium supplies the plasmid to immune cells, for example, B cells and dendritic cells, probably by passage through the intestinal barrier. High levels of protection have been established using this methodology. Such delivery methods are useful for aspects of the invention that utilize the systemic delivery of the antigen, the CpG type nucleic acid and / or other therapeutic agent.

Similar to CpG nucleic acids, the CpG type nucleic acids are useful as adjuvants to induce  a systemic immune response. In this way, acids CpG type nuclei can be supplied to an individual exposed to an antigen to produce an immune response enhanced against the antigen. The combinations of two or more different CpG type nucleic acids are also useful as adjuvants to induce a systemic immune response.

CpG type nucleic acids can also be combined with other therapeutic agents as adjuvants for boost immune responses. CpG type nucleic acid and other therapeutic agent can be administered simultaneously or sequentially When the other therapeutic agents are administered simultaneously, can be administered in it or separate formulations, but they are administered at the same time. The other therapeutic agents are administered sequentially with the others and with the CpG type nucleic acid, when the administration of the other therapeutic agents and nucleic acid CpG type is temporarily separated. The separation in time between the administration of these compounds may be a matter of minutes or it can be longer. Other therapeutic agents include, but are not limited to, adjuvants, cytokines, antibodies, antigens, etc.

CpG nucleic acids are adjuvants that they can be combined with CpG type nucleic acids of the invention to boost immune responses. Other nucleic acids immunostimulators, in addition to CpG nucleic acids and acids CpG type nucleic acids can also be combined with acids CpG type nuclei of the invention to enhance responses immune. These other immunostimulatory nucleic acids include poly-G nucleic acids and nucleic acids rich in T.

A number of references describe the immunostimulatory properties of poly-G nucleic acids. Pisetsky and Reich (1993) Mol Biol Reports 18: 217-221; Krieger and Herz (1994) Ann RevBiochem 63: 601-637; Macaya et al. (1993) Proc Natl Acad Sci USA 90: 3745-3749; Wyatt et al. (1994) Proc Natl Acad Sci USA 91: 1356-1360; Rando and Hogan (1998) in: Applied Antisense Oligonucleotide Technology, eds. Rrieg and Stein, p. 335-352; and Kirnura et al. (1994) JBiochem 116: 991-994.

The poly-G nucleic acid, in preferred embodiments, comprises one of the following formulas:

5' X_ {1} X_ {2} GGGX_ {3} X_ {4} 3',

where X_ {1}, X_ {2}, X_ {3} and X4 are nucleotides, 5 'GGGNGGG 3' or 5 'GGGNGGGNGGG 3', where N represents between 0 and 20 nucleotides. In some embodiments, at minus one of X_ {3} and X_ {4} is a G and in other embodiments, both X_ {3} and X_ {4} are Gs.

Recently, Dr. Arhur Krieg discovered that T-rich nucleic acids are immunostimulators. At the international symposium "Immunobiology of Bacterial CpG-DNA", presented in Bavaria from September 26 to 29, 1999, Dr. Krieg presented that poly-T nucleic acids 24 bases in length are immunostimulators, while poly nucleotides -C of the same length are not immunostimulators. As used herein, "T-rich nucleic acid" refers to a nucleic acid that includes at least one poly-T sequence and / or that has a nucleotide composition of more than 25 percent thymine nucleotide residues (T) . A poly-T sequence includes at least four consecutive T nucleotides and does not require the presence of a CpG motif. An A-rich nucleic acid may, optionally, be free of unmethylated CpG dinucleotides or be free of CpG dinucleotides
methylated

In addition, to combine type nucleic acids CpG with other CpG type nucleic acids or other nucleic acids adjuvant immunostimulators, the compositions of the invention they can also be administered with non-acidic adjuvants nucleic A non-nucleic acid adjuvant is any molecule or compound except CpG type nucleic acids described herein that can stimulate the humoral and / or cellular immune response. Non-nucleic acid adjuvants include, for example, adjuvants that create a store effect, immune stimulating adjuvants, and adjuvants that create a warehouse effect and stimulate the system immune

An adjuvant that creates a store effect, such as used here, it is an adjuvant that causes the antigen to be released slowly in the body, thus prolonging the exposure of the antigen to immune cells. This class of adjuvants include, but are not limited to, aluminum (for example, aluminum hydroxide, aluminum phosphate); or emulsion formulations that include mineral oil, non-mineral oil, water-in-oil emulsions or oil in water, oil in water emulsions as ISA adjuvants of the Montanida series (for example, Montanida ISA 720, AirLiquide, Paris, France); MF-59 (an emulsion squalene in water stabilized with Span 85 and Tween 80; Chiron Corporation, Emerville, CA; and PRO VAX (an oil-in-water emulsion which contains a stabilizing detergent and a forming agent micelles; IDEC, Pharmaceuticals Corporation, San Diego, CA).

An immuno-stimulant adjuvant is an adjuvant that causes activation of a cell in the immune system. For example, it could cause an immune cell to produce and secrete cytokines. This class of adjuvants includes, but is not limited to, purified saponins from the bark of the Q. saponaria tree, such as QS21 (a glycolipid eluting at the 21st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, MA); poly [di (carboxyphenoxy) phosphazene (PCPP polymer; Virus Research Institute, USA); lipopolysaccharide derivatives such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, MT), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosaline disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and the elongation factor of Leishmania (a purified protein from Leishmania ; Corixa Corporation, Seattle, WA).

The adjuvants that create a store effect and stimulate the immune system are those compounds that They have both of the functions identified above. This kind of adjuvants include, but are not limited to, ISCOMS (complexes immunostimulators containing mixed saponins, lipids and form particles the size of a virus with pores that can carry a antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham Adjuvant System # 2 which is an emulsion oil in water containing MPL and QS21: SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (a # 4 SmithKline Beecham adjuvant system containing aluminum and MPL; SBB, Belgium); nonionic block copolymers that form micelles as CRL 1005 (these contain a linear polypropylene chain hydrophobic flanked by polyoxyethylene chains; Vaxcel, Inc., Norcross, GA); and Syntex Adjuvant Formulations (SAF, a oil-in-water emulsion containing Tween 80 and a copolymer non-ionic block; Syntex Chemicals, Inc., Boulder, CO).

CpG type nucleic acids are also useful as mucosal adjuvants. It has previously been discovered that both immunities, systemic and humoral, are induced by the mucosal supply of CpG nucleic acids. Immunity Systemically induced in response to CpG nucleic acids included both responses, humoral and cell-mediated, to antigens specific, which were not able to induce systemic immunity when administered alone in the mucosa. Other, both acids CpG nuclei such as cholera toxin (CT, a mucosal adjuvant that induces a Th2 response) induces T lymphocytes cytotoxic (CTL). This was surprising, since, with immunization systemically, the presence of Th2 type antibodies is associated normally with the lack of CTL (Schirmbeck et al., 1995). Based In the results presented here, it is expected that acids CpG type nuclei work similarly.

In addition, CpG-type nucleic acids induce a mucosal response in both local mucosal sites (for example, lung) and remora (for example, lower digestive tract). Significant levels of IgA antibodies are induced at sites distant from the mucosa by CpG type nucleic acids. Generally, CT is considered to be a highly effective mucosal adjuvant. As previously demonstrated (Snider DP (1995) Crit Rev Immunol 15: 317-48), CT predominantly induces the IgG1 isotype of antibodies, which are indicative of the Th2 type response. In contrast, CpG type nucleic acids are more Th1 with predominantly IgG2a antibodies, especially after stimulation or when the two adjuvants are combined. In general, Th1 type antibodies have better stabilizing abilities, and in addition, a Th2 response in the lung is highly undesirable because it is associated with asthma. Kay AB (1996) Ann NY Acad Sci 796: 1-8; Hogg JC (1997) APMIS 105: 735-45. Therefore, the use of CpG type nucleic acids as mucosal adjuvants have benefits that other mucosal adjuvants cannot achieve. The CpG type nucleic acids of the invention are also useful as mucosal adjuvants to induce both the systemic and mucosal immune responses.

Mucosal adjuvants referred to as non-nucleic acid mucosal adjuvants can also be administered with CpG type nucleic acids. A non-nucleic acid mucosal adjuvant, as used herein, is an adjuvant other than the CpG type nucleic acid, which is capable of inducing a mucosal immune response in an individual when administered on a mucosal surface in conjunction with an antigen. Mucosal adjuvants include, but are not limited to, bacterial toxins, for example Cholera toxin (CT), derivatives of CT, which include, but are not limited to, the CT subunit B (CTB) (Wu et al., 1998, Tochikubo et al., 1998); CTD53 (Asp by Val) (Fontana et al., 1995); CTK97 (Lys by Val) (Fontana et al., 1995); CTK104 (Lys by Tyr) (Fontana et al., 1995); CTD53 / K63 (Asp by Val, Lys by Ser) (Fontana et al., 1995); CTH54 (His by Arg) (Fontana et al , 1995); CTN107 (Asn by His) (Fontana et al., 1995); CTE114 (Glu per Ser) (Fontana et al., 1995); CTE112K (Lys per Glu) (Yamamoto et al., 1997a); CTS61F (Phe por Ser) (Yamamoto et al., 1997a, 1997b); CTS106 (Lys by Pro) (Douce et al., 1997, Fontana et al., 1995); and CTK63 (Lys by Ser) (Douce et al., 1997, Fontana et al., 1995), zonula occludens toxin, zot, heat labile enterotoxin from Escherichia coli , labile toxin (LT), LT derivatives including, but not limited to, subunit B of LT (LTB) (Verweij et al., 1998); LT7K (Lys by Arg) (Komase et al., 1998, Douce et al., 1995); LT61F (Phe por Ser) (Komase et al., 1998); LT112K (Lys by Glu) (Komase et al., 1998); LT118E (Glu by Gly) (Komase et al., 1998); LT146E (Glu by Arg) (Komase et al., 1998); LT192G (Gly by Arg) (Komase et al., 1998); LTK63 (Lys por Ser) (Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al., 1996); and LTR72 (Ala to Arg) (Giuliani et al , 1998), Pertussis toxin, PT. (Lycke et al., 1992, Spangler BD, 1992, Freytag and detriments, 1999, Roberts et al., 1995, Wilson et al., 1995) including PT-9K / 129G (Roberts et al., 1995, Cropley et al. ., nineteen ninety five); toxin derivatives (see below) (Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al., 1995, Freytag and Clements, 1999); lipid A derivatives (eg, monophosphoryl lipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998; muramyl dipeptide derivatives (MDP) (Fukushima et al., 1996, Ogawa et al., 1989 , Michalek et al., 1983, Morisaki et al., 1983); bacteria outer membrane proteins (eg, outer surface protein A (OspA) lipoprotein from Borrelia burgdorferi, outer membrane protein from Neisseria meningitidis ) (Marinaro et al., 1999, Van de Verg et al., 1996); oil-in-water emulsions (eg, MF59) (Barchfield et al., 1999, Verschoor et al., 1999, O'Hagan, 1998); aluminum salts (Isaka et al., 1998, 1999); and saponins (eg, QS21, Aquila Biopharmaceuticals, Inc., Worcester, MA) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMS, MF -59 (an emulsion of squalene in water stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, CA); Seppic ISA series of Montanide adjuvants (for example, Montanide ISA 720; A irLiquide, Paris, France); PRO VAX (an oil-in-water emulsion containing a detergent and a micelle forming agent; EDEC Pharmaceuticals Corporation, San Diego, CA); Syntext Adjuvant Formulation (SAF; Syntex Chemicals, Inc., Boulder, CO);

poly [di (carboxylatenophene) phosfacene (PCPP polymer; Virus Research Institute, USA) and the factor of elongation of Leishmania (Corixa Corporation, Seattle, WA).

Immune responses can also be induced or enhanced by co-administration or co-linear cytokine expression (Bueler and Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki et al., 1997; Kim et al., 1997), or by co-stimulatory molecules (eg, B7-1 (CD20), B7-2 (CD86); Iwasaki et al., 1997; Tsuji et al., 1997) with nucleic acids CpG type. CpG type nucleic acids can also be formulated or administered to an individual in conjunction with a cytokine. The cytokines can be administered directly with the CpG type nucleic acids or they can be administered in the form of nucleic acid vectors encoding the cytokine, so that the cytokine can be expressed in vivo. In one embodiment, the cytokine is administered in the form of a plasmid expression vector.

       \ global \ parskip1.000000 \ baselineskip
    

The term "cytokine" is used as a generic name for a diverse group of peptides and proteins soluble that act as humoral regulators at concentrations of nanomolar to picomolar and that, both in normal conditions and pathological, modulate the functional activities of cells individual and tissue. These proteins also mediate directly the interactions between cells and regulate processes that They take place in the extracellular environment. Examples of cytokines include, but are not limited to, interleukin (IL) -1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, stimulating factor of granulocyte-macrophage colonies (GM-CDF), colony stimulating factor of granulocytes (G-CSF), interferon-? (IFN-?), IFN-?, Tumor necrosis factor (TNF) -?, Transforming growth factor (TGF) -?, Flt3 ligand, and CD40 ligand. In certain preferred embodiments, the cytokine that is formulated or is administered to an individual in conjunction with an acid CpG type nucleic is selected from IL-2, IL-3, IL-4, IL-18, IFN-?, IFN-?, TNF-?, Flt3 ligand, G-CSF, and GM-CSF. In certain embodiments where the CpG type nucleic acid is administered to an individual in an amount effective to induce a cytokine as an induction aspect of an immune response, the Induced cytokine may include any of the following: IL-1?, IL-2, IL-6, IL-12, IL-18, TNF-?, IFN-?, E IFN- γ.

Cytokines play a role directing the T cell response Helper T cells (CD4 +) orchestrate the mammalian immune response through the production of soluble factors that act on other system cells immune, including other T cells. Many of the cells Mature T helper CD4 + express one or two cytokine profiles: Th1 or Th2. Th1 cytokines characteristically include IFN- γ, IL-12, e IL-2 The Th1 set promotes hypersensitivity delayed type, cell-mediated immunity, and the Immunoglobulin class exchange to IgG2a. Conversely, Th2 cytokine profile characteristically includes IL-4, IL-5, IL-10 e IL-13 The Th2 set induces humoral immunity by activation of B cells, promoting the production of antibodies, and inducing class exchange to IgG1 and IgE. In some embodiments, it is preferred that the cytokine be a Th1 cytokine.

The nucleic acids of the invention are also useful for redirecting an immune response from a Th2 immune response to a Th1 immune response. The redirection of an immune response from a response Th2 to Th1 immune can be assessed by measuring the levels of cytokines produced in response to nucleic acid (for example, inducing monocytic cells and other cells to produce  Th1 cytokines, which include IL-12, IFN- γ and GM-CSF). The redirection of the immune response from a Th2 response to Th1 is particularly useful for the treatment or prevention of asthma. For example, an effective amount of a nucleic acid of the type CpG to treat asthma, that amount can be useful to redirect an immune response of type Th2, which is associated with the asthma, to a response of type Th1. Th2 cytokines, especially IL-4 and IL-5, are elevated in the airways of asthmatic individuals. These cytokines promote important aspects in the response asthmatic inflammatory, which include the exchange of the IgE isotype, eosinophilic chemotaxis, and cell activation and degranulation Mastocytic Th1 cytokines, especially the IFN- γ E IL-12, can suppress Th2 clone formation and cytokine production Th2.

The nucleic acids of the invention can be formulated with or administered to an individual with an agent anti-microbial An agent anti-microbial, refers to a compound that is given naturally, semi-synthetic, or synthetic, which is capable of killing or inhibiting infectious microorganisms. The kind of useful anti-microbial agent, according to the invention will depend on the type of microorganism with which the individual is infected or at risk of being infected. The anti-microbial agents include, but not limited to, antibacterial agents, antiviral agents, agents antifungals and antiparasitic agents.

A type of agent Anti-microbial is an antibacterial agent. The antibacterial agents kill or inhibit growth or function of bacteria A large class of antibacterial agents are the antibiotics Antibiotics, which are effective in killing or inhibit a wide range of bacteria, refer to antibiotics  Broad spectrum Other types of antibiotics are predominantly effective against class bacteria Gram-positive or Gram-negative. These types of antibiotics refer to spectrum antibiotics. narrow. Other antibiotics that are effective against only one organism or disease and not against other types of bacteria, it refer to antibiotics of limited spectrum.

Sometimes the antibacterial agents are classified based on their main mode of action. In general, antibacterial agents are inhibitors of the synthesis of cell wall, cell membrane inhibitors, inhibitors of protein synthesis, inhibitors of function or synthesis of nucleic acids, and competitive inhibitors. Inhibitors of the synthesis of the cell wall inhibit the passage of the process of synthesis of the cell wall, and, in general, the synthesis of bacterial peptidoglycan. The inhibitors of the synthesis of cell wall include antibiotics β-lactams, natural penicillins, semi-synthetic penicillins, ampicillin, acid clavulanic, cephalosporins, and bacitracin.

Β-lactams are antibiotics that contain a β-lactam member ring, which inhibits the last step of peptidoglycan synthesis. The β-lactam antibiotics can be synthetic or natural. Natural antibiotics are generally produced by two groups of fungi, the molds of Penicillium and Cephalosporium . The β-lactam antibiotics produced by Penicillium are natural penicillins, such as penicillin G or penicillin V. These are produced by fermentation of Penicillium Chrysogenum . Natural penicillins have a narrow spectrum of activity and are generally effective against Streptococcus, Gonococcus, and Staphylococcus. Other types of natural penicillins, which are also effective against Gram-positive bacteria, include penicillins F, X, K, and O.

Semi-synthetic penicillins they are generally modifications of the acid molecule 6-aminopenicillanic produced by a mold. Acid 6-aminopenicillanic can be modified by addition of side chains, which produce penicillins that have spectra of broader activity than natural or other penicillins advantageous properties. Some types of penicillins semi-synthetics have a broad spectrum compared to Gram-positive bacteria and Gram-negative, but are inactivated by penicillinase Semi-synthetic penicillins include ampicillin, azlocillin, carbenicillin, mezlocillin, oxacillin, and piperacillin. Other types of penicillins semi-synthetic have narrower activities against Gram-positive bacteria, but they have developed properties like that are disabled by penicillinase These include, for example, dicloxacillin, methicillin, and naphcillin. Some of the penicillins semi-synthetic broad spectrum can be used in combination with inhibitors of β-lactamase, such as clavranic acids and sulbactam Β-lactamase inhibitors they have no antimicrobial action, but they work to inhibit the penicillinase, thereby protecting penicillin semi-synthetic degradation.

Some of the serious side effects associated with penicillins, both natural and semi-synthetic, are penicillin allergies. The Penicillin allergies are very serious and can cause death quickly. In an individual allergic to penicillin, the molecule of β-lactam will attack a whey protein that  initiates the IgE-mediated immune response. The answer Immune inflammatory leads to anaphylaxis and possibly to death.

Another type of β-lactam antibiotic is cephalosporin. Cephalosporins are produced by Cephalolsporium molds, and have a penicillin-like form of action. They are sensitive to degradation by bacterial β-lactamases, and therefore, they alone are not always effective. However, cephalosporins are resistant to penicillinase. They are effective against a variety of Gram-positive and Gram-negative bacteria. Cephalosporins include, but are not limited to, Cefaclor, cefamandol, cefazolin, cefetamet, cefixime, cefoperazone, cefotaxime, cefoxitin, cefsulodine, ceftacidine, ceftriaxone, cefuroxin, cephalexin, cephalothin, cefapirin.

Bacitracin is another type of antibiotics that inhibit cell wall synthesis. These antibiotics, produced by Bacillus spices, prevent cell wall growth by inhibiting the release of muropeptide or peptidoglycan subunits from the molecule from which the subunit to the outside of the membrane is derived. Although bacitracin is effective against Gram-positive bacteria, its use is generally limited to topical administration due to its high toxicity. Since lower effective doses of bacitracin can be used when the compound is administered with the immunostimulatory nucleic acids of the invention, this compound can be used systematically and the toxicity can be reduced.

Carbapenem is another broad-spectrum β-lactam antibiotic, which is capable of inhibiting cell wall synthesis. Examples of carbapenem include, but are not limited to, imipenem. Monobactams are also broad-spectrum β-lactam antibiotics, and include aztreonam. An antibiotic produced by Streptomyces , vancomycin, is also effective against Gram-positive bacteria to inhibit cell membrane synthesis.

Another class of antibacterial agents are the antibacterial agents that are cell membrane inhibitors. These compounds disorganize the structure or inhibit the function of Bacterial membranes Membrane alteration cytoplasmic bacteria results in the escape of cell material of the cell. Compounds that inhibit or interfere with the cell membrane cause cell death because the integrity of cytoplasmic membranes and External is vital for bacteria. A problem with the agents antibacterials that are cell membrane inhibitors is that they can produce effects in eukaryotic cells as well as in bacteria, due to the similarity of phospholipids of bacterial and eukaryotic membranes. Therefore, these compounds, they are rarely specific enough to allow These compounds are used systematically and prevent the use of high doses for local administration.

A clinically useful antibacterial agent that is a cell membrane inhibitor is Polymyxin, produced by Bacillus polymyxis . Polymyxins interfere with membrane function by binding to membrane phospholipids. Mostly, polymyxin is effective against Gram-negative bacteria and is generally used in severe Pseudimonas infections or in Pseudomonas infections that are resistant to less toxic antibiotics. It is also used in some limited topical examples. Its limited use is due to the severe side effects associated with systemic administration, such as damage to the kidney and other organs.

Other cell membrane inhibitors include Amphotericin B and Nystatin, produced by the Streptomyces bacteria, which are also antifungal agents, predominantly used in the treatment of systemic fungal infections and in Candida yeast infections , respectively. Imidazoles, produced by the Streptomyces bacteria, are another class of antibiotics that are cell membrane inhibitors. Imidazoles are used as antibacterial agents as well as antifungal agents, for example, used for the treatment of yeast infections, dermatophytic infections, and systemic fungal infections. Imidazoles include, but are not limited to, clotrimazole, fluconazole, itraconazole, ketoconazole, and miconazole.

Many antibacterial agents are inhibitors of protein synthesis. These compounds prevent the bacterium from synthesizing structural proteins and enzymes, and therefore, cause the inhibition of bacterial cell growth or function or cell death. In general, these compounds interfere with the transcription or translation processes. Antibacterial agents that block transcription include, but are not limited to, Rifampins, produced by the Streptomyces bacteria, and Etambutol, a synthetic chemical. Rifampins, which inhibit the RNA polymerase enzyme, have a broad spectrum or activity and are effective against Gram-positive and Gram-negative bacteria, just as Etambutol is effective against Mycobacterium tuberculosis.

Antibacterial agents that block the translation, interfere with bacterial ribosomes to prevent that mRNA be translated into proteins. In general, this kind of Compounds include, but are not limited to, tetracyclines, chloramphenicol, macrolides (for example, azithromycin, erythromycin) and aminoglycosides (for example, amikacin, gentamicin, kanamycin, streptomycin, tobramycin).

Some of these compounds bind reversibly to the 30S ribosomal subunit and cause poor mRNA reading, for example, aminoglycosides. Aminoglycosides are a class of antibiotics that are produced by the Streptomyces bacteria, such as, for example, amikacin, gentamicin, kanamycin, streptomycin, and tobramycin. Aminoglycosides have been used against a wide variety of bacterial infections caused by Gram-positive and Gram-negative bacteria. Streptomycin has been widely used as the main medicine in the treatment of tuberculosis. Gentamicin is used against many strains of Gram-positive and Gram-negative bacteria, including infections with Pseudomonas , especially in combination with tobramycin. Kanamycin is used against many Gram-positive bacteria, including penicillin-resistant Staphylococci . A side effect of aminoglycosides, which has clinically limited their use, is that at doses that are essential for their effectiveness,
It has been seen that prolonged use damages renal function and causes damage to the auditory nerves, leading to deafness.

Another type of antibacterial agent that inhibits translation is tetracyclines. Tetracyclines bind reversibly to the 30S ribosomal subunit and interfere with the binding of the tRNA loaded with the bacterial ribosome. Tetracyclines are a class of antibiotics, produced by the Streptomyces bacteria, which are broad spectrum and are effective against a variety of Gram-positive and Gram-negative bacteria. Examples of tetracyclines include chlortetracycline, doxycycline, monocycline, and tetracycline. These are important for the treatment of many types of bacteria, but they are particularly important in the treatment of Lyme disease ( Borrelia burgdorferi ).

As a result of its low toxicity and effects minimal side effects, tetracyclines have been widely used and misused in the medical community, generating problems. For example, its excessive use has led to the development of resistance extended. When used in combination with nucleic acids immunostimulators of the invention, these problems can be minimized and tetracyclines can be used effectively to The broad spectrum of treatment of many bacteria.

Antibacterial agents, such as macrolides, bind reversibly to the 50S ribosomal subunit and inhibit the elongation of the protein by peptidyl transferase, or prevent the release of uncharged tRNA from the bacterial ribosome, or both. Macrolides contain many lactone rings linked through glycoside bonds with amino sugars. These compounds include azithromycin, clarithromycin, clindamycin, erythromycin, lincomycin, oleadomycin, and roxithromycin. Erythromycin is active against most Gram-positive, Neisseria, Legionella and Haemophilus bacteria , but not against Enter obacteriaceae. Lincomycin and clindamycin, which block the formation of the peptide bond during protein synthesis, are used against Gram-positive bacteria.

Another type of translation inhibitor is chloramphenicol. Chloramphenicol binds to ribosome 70S, inhibiting the bacterial enzyme peptidyl transferase and thereby preventing the growth of the polypeptide chain during protein synthesis. Chloramphenicol can be prepared from Streptomyces or it can be entirely produced by chemical synthesis. A serious side effect associated with chloramphenicol is aplastic anemia. Aplastic anemia develops at doses of chloramphenicol that are effective in treating bacteria in small proportions (1 / 50,000) of patients. Once the antibiotic is highly prescribed, chloramphenicol is rarely used due to anemia deaths. Due to its effectiveness, it is still used in life threatening situations (for example, typhoid fever). By combining chloramphenicol with the immunostimulatory nucleic acids, the compounds can, again, be used as antibacterial agents, since the immunostimulatory agents allow the use of a lower dose of chloramphenicol, a dose that does not produce side effects.

Some antibacterial agents interrupt the synthesis or function of nucleic acids, for example, the binding of DNA or RNA, so that their messages cannot be read. These include, but are not limited to, quinolones and co-trimazole, both synthetic chemicals, and rifamucines, a natural or semi-synthetic chemical. Quinolones block bacterial DNA replication by inhibiting DNA gyrase, the enzyme needed by the bacteria to produce its circular DNA. They are broad spectrum and examples include ciprofoxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, and temafloxacin. Nalidixic acid is an antibacterial agent that binds to the enzyme DNA gyrase (topoisomerase), which is essential for DNA replication, and allows super-spirals to be relaxed and reformed, inhibiting the activity of DNA gyrase. The main use of nalidixic acid is in the treatment of lower urinary tract infections (UTI), because it is effective against several types of Gram-negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae, and Proteus, species which are the common cause of UTI. Co-trimazole is a combination of sulfamethoxazole and trimethoprim, which blocks the bacterial synthesis of folic acid, necessary to make DNA nucleotides. Rifampicin is a derivative of rifamycin that is active against Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis caused by Neisseria meningitidis ) and some Gram-negative bacteria. Rifampicin binds to the beta subunit of the polymerase and blocks the addition of the first nucleotide, which is necessary to activate the polymerase, and therefore, blocks mRNA synthesis.

Another class of antibacterial agents are compounds that function as competitive inhibitors of bacterial enzymes. Competitive inhibitors are all mostly structurally similar to the bacterial growth factor and compete to bind, but do not carry out the metabolic function in the cell. These compounds include sulfonamides and chemically modified forms of sulfomanides that even have greater and wider antibacterial activity. Sulfonamides (for example, gastrisin and trimethoprim) are useful for the treatment of Streptococcus pneumoniae, Streptococci beta-hemolytic and E. coli, and have been used in the treatment of uncomplicated UTIs caused by E. Coli , and in the treatment of meningococcal meningitis.

Antiviral agents are compounds that prevent infection of cells by virus or the replication of virus inside the cell. There are fewer antiviral medications that antibacterial drugs, because the process of viral replication is closely related to DNA replication inside the host cell, so the antiviral agents sometimes not specific, they could be toxic to the host.

There are several scenarios within the process of viral infection that can be blocked or inhibited by agents antivirals These scenarios include coupling the virus to the host cell (immunoglobulin or binding peptides), virus decapsulation (for example, amantadine), synthesis or translation of viral mRNA (for example, interferon), replication of the RNA or viral DNA (eg nucleoside analogs), maturation of new viral proteins (for example, protease inhibitors), and virus preparation and release.

Nucleotide analogs are compounds synthetics that are similar to nucleotides, but that have a deoxyribose or incomplete or abnormal ribose group. Once the nucleotide analogs are in the cell, they phosphorylate, producing the triphosphate form that competes with nucleotides normal for incorporation into ADR or viral RNA. Once the Triphosphate form of the nucleotide analog is incorporated into of the growing nucleic acid chain, cause association irreversible with viral polymerase and with it, the termination of chain. Analog nucleotides include, but are not limited to, acyclovir (used to treat herpes simplex virus and varicella zoster virus), ganciclovir (useful for treatment of cytomegalovirus), idoxuridine, ribavirin (useful for treatment of respiratory syncytial virus), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymine).

Interferons are cytokines that are secreted by virus-infected cells, as well as by immune cells Interferons work by receptor binding specific on cells adjacent to infected cells, causing the change in the cell that protects it from infection by the virus. Interferons? And? Also induce expression of MHC class I and II molecules on the surface of infected cells, resulting in an increase in antigen presentation for immune cell recognition host Interferons α and β are available as recombinant forms and have been used for treatment of chronic hepatitis B and C infection. At doses that are effective for entiviral therapy, interferons have severe effects side effects such as fever, malaise and weight loss.

Immunoglobulin therapy is used. for the prevention of viral infection. Therapy with immunoglobulins for viral infections is distinct from therapy with immunoglobulins for bacterial infections, because, more than be specific antigen, immunoglobulin therapy works by binding to extracellular virions and prevents them from being attacked and entering cells that are susceptible to viral infection. The therapy is useful for the prevention of viral infection during period of time that the antibody is presented in the host. In general, there are two types of immunoglobulin therapies, therapy normal immune globulin and globulin therapy hyper-immune Normal immune therapy with globulin uses an antibody product that is prepared from whey of normal blood donors and that is grouped. This product Clustered contains low antibody titers from a wide range of human viruses, such as hepatitis A, parvovirus, enterovirus (especially in neonates). Hyper-immune therapy globulin uses antibodies that are prepared from serum of individuals who have high titers of an antibody of a virus in particular. These antibodies are used against a virus specific. Examples of hyper-immune globulins include zoster immune globulins (useful for the prevention of chickenpox in children and immunocompromised infants), immunoglobulin of human rabies (useful in prophylaxis post-exposure of an individual bitten by a rabid animal), hepatitis B immune globulin (useful for hepatitis B virus prevention, especially in a individual exposed to the virus), and RSV immune globulin (useful in the treatment of respiratory syncytial virus infections).

Another type of immunoglobulin therapy is the passive immunization This involves the administration of antibodies or fragments of viral surface proteins. Two types of vaccines that are available for passive immunization for hepatitis B include hepatitis B antibodies derived from recombinant hepatitis B serum and antibodies. Both are prepared from hepatitis B surface antigen (HBsAg). The antibodies are administered in three doses at individuals at high risk of infection with the hepatitis virus  B, as health center workers, sexual partners of chronic carriers, and children.

Antifungal agents are useful for Treatment and prevention of infectious fungi. The agents Infectious, they are sometimes classified by their mechanism of action. Some infectious agents function as wall inhibitors cell inhibiting glucose synthase. These include, but I don't know limited to, basiungin / ECB. Other antifungal agents work destabilizing the integrity of the membrane. These include, but are not limited to, imidazoles, such as clotrimazole, fluconazole, itraconazole, ketoconazole, miconazole, sertaconzole, and voriconazole, as well as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimycin, UK 292, butenafme, and terbinafme. Other agents Antifungals work by breaking chitin (for example, chitinase) or by immunosuppression (cream 501). Some examples of agents Commercially available antifungals are shown in the Table one.

       \ vskip1.000000 \ baselineskip
    

TABLE 1 Antifungal Agents

one

Examples of antiparasitic agents, also called parasiticides, useful for human administration, include, but are not limited to, albendazole, amphotericin B, benznidazole, bitionol, chloroquine HC1, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, doxycycline, eflornithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, Meglumine antimony, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquina, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethamine-sulfadoxino, pyrimethamine sulfonamides, quinacrine HC1, quinine sulfate, quinidine gluconate, spiramycin, stibogluconata sodium (sodium gluconate antimony), suramin, tetracycline, thiabendazole, tinidazole, trimetroprim-sulfamethoxazole, and triparsamide, some of which are used alone or in combination with others.

Parasiticides used in non-human individuals include albendazole, amprolium; decoquinate, benzimidazoles such as fenbendazole, clorsulon, dichlorvos, diethylcarbamazine, doramectic, epsiprantel, febantel, fenbendazole, hygromycin B, sodium tiacetarsemida, iprinomectina, ivermectinaa, lasalocid, levamisole, melarsomine, metronidazole, milbemycin oxime, monensin sulfadimethoxine, moxidectin, N-butyl chloride , oxfendazole, oxybenzole, piperazine, praziquantel, pyrantel pamoate, pyrantel tartrate, sulfametazine, sulfaquinoxaline, thiabendazole, and toluene. Itulized parasiticides in horses include mebendazole, oxfendazole, febantel, pyrantel, dichlorvos, trichlorfon, ivermectin, piperazine; for S. westeri: ivermectin, benzimidazoles such as thiabendazole, cambendazole, oxibendazole and fenbendazole. Useful parasiticides in dogs include milbemycin oxine, ivermectin, pyrantel pamoate and the combination of ivermectin and pyrantel. Treatment of parasites in creeds may include the use of levamisole, piperazine, pyrantel, thiabendazole, dichlorvos and fenbendazole. In sheep and goats, anthelmintic agents include levamisole or ivermectin. Caparsolate has shown some efficacy in the treatment of D. immitis (heartworm) in cats.

CpG type nucleic acids can also be formulated with or administered to an individual in conjunction with an anti-allergic or anti asthmatic therapy. In certain embodiments, an asthma / allergy medication is a medicine selected from the group consisting of inhibitor of phosphodiesterase (PDE) -4, Bronchodilator / beta-2 agonist, channel opener of K +, antagonist VLA-4, antagonist of Neurokina, TXA2 synthesis inhibitor, Xantanine, acid antagonist Arachidonic, 5 lipoxygenase inhibitor, antagonist of Thromboxin A2 receptor, Thromboxane A2 antagonist, 5-lipox activation protein inhibitor, e Protease inhibitor, but not so limited. In some important embodiments, the asthma / allergy medication in a Bronchodilator / beta-2 agonist selected from group consisting of salmeterol, salbutamol, terbutaline, D2522 / formoterol, fenoterol, and orciprenaline.

In another embodiment, the medication for asthma / allergy is a medication selected from the group consisting of in Anti-histamines and inductors of Prostaglandins. In one embodiment, the anti-histamine is selected from the group consisting of astemizole, azelastine, betatastine, buclizine, cetirizine, cetirizine analogues, CS 560, desloratadine, ebastine, epinastine, fexofenadine, HSR 609, levocabastine, loratidine, mizolastine, norastemizole, terfenadine, and tranilast. In another embodiment, the Prostaglandin inducer is S-5751.

Still, in another embodiment, the medication for asthma / allergy is selected from the group consisting of Steroids and immunomodulators. The immunomodulators can be selected from the group consisting of agents anti-inflammatory, leukotriene antagonists, IL-4 muteins, soluble receptors of IL-4, Immunosuppressants, antibodies anti-IL-4 antagonists anti-IL-4 antibodies anti-IL-5, Fc fusion proteins of the soluble EL-13 receptor, antibodies anti-IL-9, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, and IgE downregulators, but not so limited. In one embodiment, In IgE donwregulator is an anti-IgE.

In another embodiment, the steroid is selected from the group consisting of beclomethasone, budesonide, fluticasone, methylprednisolone, prednisone, and triamcinolone. Also, in a further implementation, the immunosuppressant is a vaccine of the Tolerizing peptide.

CpG type nucleic acids can also be formulated with or administered to an individual in conjunction with An anti-cancer therapy. Therapies anti-cancer drugs include Cancer, radiation and surgical procedures. As used here, a "cancer medicine" refers to an agent pharmacist who is administered to an individual for the purpose of treat cancer As used here, "treat cancer" includes preventing the development of cancer, reducing the symptoms of cancer, and / or inhibit the growth of an established cancer. In other aspects, the cancer drug is administered to a individual at risk of developing cancer, for the purpose of Reduce the risk of developing cancer. Several are described here types of medications to treat cancer. For the purpose of this specification, cancer medications are classified as chemotherapeutic agents, agents immunotherapeutics, radiotherapeutic agents, vaccines against cancer, hormone therapy, and response modifiers biological

In addition, the methods of the invention are intended cover the use of more than one cancer drug along with CpG type nucleic acids. As an example, whenever appropriate, CpG type nucleic acids can be administered with both agents, both chemotherapeutic and immunotherapeutic Alternatively, the medication for the cancer may include an immunotherapeutic agent and a vaccine against cancer, or a chemotherapeutic agent and a vaccine against cancer, or a chemotherapeutic agent, an immunotherapeutic agent and a cancer vaccine, all administered to an individual as the purpose of treating the individual who has cancer or with risk of developing cancer.

Cancer medications work in a variety of ways. Some anti-cancer medications work by targeting physiological mechanisms that are specific to tumor cells. Examples include, targeting, specific genes (and their gene products, that is, primarily proteins) that are mutated in cancer. Such genes include, but are not limited to, oncogenes (e.g., Ras, Her2, blc-2), tumor suppressor genes (e.g., EGF, p53, Kb), and cell cycle targets (e.g., CDK4, p21, telomerase). Cancer drugs, alternatively, target signal transduction pathways and molecular mechanisms that are altered in cancer cells. The targeting of cancer cells, via epitopes expressed on their cell surface, is achieved through the use of monoclonal antibodies and derivatives and analogues of monoclonal antibodies. The latter type of cancer medications are generally referred to here as a form of immunotherapy. Immunotherapies also include cytokines such as IFN-?, IL-2, and
IL-12

Other medicines for cancer, mark as target different cells of cancer cells. For example, some medications prepare the immune system to attack tumor cells (i.e. cancer vaccines). further other medicines, called angiogenesis inhibitors, They work by attacking the blood supply of solid tumors. Because malignant cancers are able to metastasize (it is that is, they leave the main tumor and settles in non-tissue adjacent, thus forming a secondary tumor), the medications that prevent this metastasis are also useful in the treatment of Cancer. Angiogenic mediators include growth factor of basic fibroblasts (b-FGF), factor of vascular endothelial growth (VEGF), angiopoietin, angiostatin, endostatin, tumor necrosis factor (TNF) -?, TNP-470, thrombospondin-1, platelet factor 4, carboxyamido-triazole (CAI), and certain members of the integrin family of proteins. Such a category of medication is a metalloprotease inhibitor, which inhibits the enzyme used by cancer to exit the main tumor and extravased to other tissue.

Some cancer cells are antigenic, with what can be the target of the immune system. In one aspect, the combined administration of CpG type nucleic acids and cancer medications, particularly those that are classified as immunotherapy against cancer, it is useful for stimulate a specific immune response against an antigen cancerous.

The theory of immune surveillance is that, a main function of the immune system is to detect and Remove neoplastic cells before the tumor forms. A basic principle of this theory is that cancer cells are antigenically different from normal cells and therefore, cause immune reactions similar to those caused by rejection of immunologically incompatible (non-self) allografts.  Studies have shaped that tumor cells differ, both qualitatively as quantitatively, in its expression of antigens For example, "tumor specific antigens" they are antigens specifically associated with tumor cells, but Not to normal cells. Examples of tumor specific antigens they are viral antigens in tumors induced by DNA or RNA viruses, as well as neo-antigens that arise from the joint abnormal genes. The "tumor associated" antigens are present in both tumor cells and normal cells, but they are present in different quantity or in a different way in tumor cells. Examples of such antigens are antigens oncofetals (eg carcinoembryonic antigen), antigens of differentiation (for example, T and Tn antigens), and products oncogenic (for example, HER / neu).

Different types of cells that can kill tumor targets have been identified in vitro and in vivo : natural killer cells (NK cells), cytolytic T lymphocytes (CLTs), lymphokine activated killer cells (LAKs), and activated macrophages. NK cells can kill tumor cells without having previously been sensitized with specific antigens, and the activity does not require the presence of class I antigens encoded by the major histocompatibility complex (MHC) of the target cells. It is thought that NK cells participate in the control of nascent tumors and in the control of metastatic growth. Unlike NK cells, CTLs can kill tumor cells only after they have been sensitized with tumor antigens and when the target antigen is expressed in the tumor cell, which also expresses MHC class I. CTLs are thought to be cells. effectors in the rejection of transplanted tumors and tumors caused by DNA viruses. LAK cells are a subset of null lymphocytes other than the NK and CTL populations. Activated macrophages can kill tumor cells in a way that is neither antigen dependent nor restricted to MHC, once activated. Activated macrophages are thought to decrease the growth rate of the infiltrating tumors. In vivo assays have identified other immune mechanisms such as antibody-dependent, cell-mediated cytotoxic reactions (ADCC) and antibody lysis plus complement. However, these effector immune mechanisms are thought to be less important than the function of NK, CTLs, LAK and macrophages in vivo (for review, see Piessens, WF, and David, J., "Tumor Immunology", In: Scientific American Medicine, Vol. 2, Scientific American Books , NY, pp. 1-13, 1996).

The goal of immunotherapy is to increase the The patient's immune response to an established tumor. A Immunotherapy method includes the use of adjuvants. The substances adjuvants derived from microorganisms, such as Bacillus Calmette-Guerin (BCG), increase the response immune and enhance tumor resistance in animals.

Immunotherapeutic agents are drugs that are derived from antibodies or antibody fragments that specifically bind or recognize a cancerous antigen. Preferably, the antibody or antibody fragment is derived from a monoclonal antibody that is specific for a tumor cell or a tumor cell antigen. Monoclonal antibodies are well known in the state of the art, and are a variety of molecules derived from monoclonal antibodies, including chimeric, humanized, and very specific monoclonal antibodies, Fab fragments, (Fab ') 2 fragments, Fv fragments, molecules Fv side chain, etc. As described above, a cancerous antigen, as used herein, is broadly defined as an antigen expressed by a cancer cell. Preferably, the antigen is expressed on the cell surface of the cancer cell. More preferably, the antigen is one that is not expressed in normal cells, or at least, not expressed at the same level as in cancer cells. Antibody-based immunotherapies can work by binding to the cell surface of a cancer cell and thereby stimulating the endogenous immune system to attack the cancer cell. Another way of working with antibody-based therapy is as an administration system for the specific labeling of toxic substances in cancer cells. In this regard, antibodies are usually conjugated with toxins such as ricin (for example, from castor seeds), calicheamycin and maitansinoids, to radioactive isotopes such as Yodo-131 (131 I) and Itrium-90 (^ 90} Y), to chemotherapeutic agents (as described herein), or to biological response modifiers. In this way, toxic substances can be concentrated in the cancer region and non-specific toxicity in normal cells can be minimized. In addition to the use of antibodies that are specific for cancerous antigens, antibodies that bind to the vascular system, such as those that bind to endothelial cells, are also useful in the invention. This is because solid tumors are generally dependent on the formation of new blood vessels for survival, and therefore, many of the tumors are able to recruit and stimulate the growth of new blood vessels. As a result, a strategy of many of the anti-cancer drugs is to attack the blood vessels that feed a tumor and / or the connective tissues (or stroma) that support these vessels
blood.

CpG type nucleic acids in conjunction with immunotherapeutic agents such as monoclonal antibodies, they are capable of increasing long-term survival through a number of mechanisms that include significant potentiation of ADCC (as discussed above), activation of NK cells, and a increase in IFN-? levels. It is believed that IFN-? induces cell activation NK Nucleic acids, when used in combination with monoclonal antibodies, serve to reduce the dose of antibody required to achieve a biological result.

Examples of cancer immunotherapies, which are currently being used, are developed in the
Table 2.

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

TABLE 2 Immunotherapies against cancer in development or in the market

2

3

4

5

6

Cancer vaccines are medications which aim to stimulate an endogenous immune response against cancer cells The current vaccines produced, predominantly activate the humoral immune system (it is ie, antibody dependent immune response). Other vaccines, currently in development, are focused on the cell-mediated immune system activation, including cytotoxic T lymphocytes, which are capable of killing cells Tumor Vaccines against cancer generally enhance the presentation of cancerous antigens to both APCs (for example, macrophages and dendritic cells) and / or other immune cells such as T cells, B cells and NK cells.

Although cancer vaccines can be of various forms, as discussed above, their purpose is to show APCs the cancerous antigens and / or cancer associated antigens, in order to facilitate endogenous processing of said antigens by APCs and the final presentation of the antigen on the cell surface in the context of MHC class I molecules. One form of cancer vaccine is a whole cell vaccine, which is a preparation of cancer cells that have been removed from a individual, have been treated ex vivo and then have been reintroduced as total cells in the individual. Lysates of tumor cells can also be used as a cancer vaccine to obtain an immune response. Another form of cancer vaccine is a peptide vaccine that uses small cancer-specific or cancer-associated proteins to activate T cells. Cancer-associated proteins are proteins that are not expressed exclusively in cancer cells (i.e., other normal cells). can still express these antigens). However, the expression of antigens associated with cancer is, generally and consistently, upregulated with cancers of a particular type.

Still another form of cancer vaccine is the dendritic cell vaccine, which includes complete dendritic cells, which have been exposed to a cancerous antigen or an antigen associated with cancer in vitro . Lysates or dendritic cell membrane fractions can also be used as cancer vaccines. Dendritic cell vaccines are capable of directly activating APCs. Other cancer vaccines include ganglioside vaccines, heat shock protein vaccines, viral and bacterial vaccines, and nucleic acid vaccines.

The use of CpG type nucleic acids together with cancer vaccines, they provide a humoral immune response specific to antigen and mediated by enhanced cell, in addition to activating NK cells and cells endogenous dendritic, and increase levels of IFN-? This empowerment allows you to use a vaccine with a reduced dose of antigen to reach the same beneficial effect that would be achieved using doses without acid CpG type nucleic. In some examples, vaccines against Cancer can be used together with adjuvants, such as those above described.

Other vaccines take the form of dendritic cells that have been exposed to cancerous antigens in vitro , have been processed and are capable of expressing cancerous antigens on their cell surface in the context of MHC molecules, for effective presentation of the antigen to other cells. of the immune system.

CpG type nucleic acids are used, in an aspect of the invention, together with vaccines against cancer, which are based on dendritic cells. A cell Dendritic is a professional antigen presenting cell. The dendritic cells form a junction between the immune system innate and acquired, presenting antigens and through their expression of a pattern of recognition receptors that detect microbial molecules such as lipopolysaccharide (LPS) in your environment local. In an effective way, dendritic cells internalize, process, and present soluble specific antigens that are exposed. The process of internalization and presentation of antigen causes rapid upregulation of complex expression of major histocompatibility (MHC) and costimulatory molecules, cytokine production, and migration to organs lymphatics, where they are believed to be involved in the activation of T cells.

Table 3 shows a variety of vaccines against cancer that are currently being used or are in developing.

       \ vskip1.000000 \ baselineskip
    

TABLE 3 Vaccines against developing or in cancer market

7

8

As used here, agents chemotherapeutic agents (and, equivalently, the agents of chemotherapy) cover all forms of anti-drug medications cancer that does not fall into agent categories immunotherapeutics or cancer vaccines. The agents chemotherapeutics, as used herein, encompass both Chemical agents as biological. These agents work by inhibiting the cellular activity in which the cancer cell in dependent To continue surviving. The categories of agents Chemotherapeutic agents include alkylating agents / alkaloids, antimetabolites, hormones or hormone analogues, and a miscellaneous of antineoplastic medications. Many, if not all, of these Agents are directly toxic to cancer cells and not They require immune stimulation. The combination of chemotherapy and Immunostimulatory nucleic acid administration increases the maximum tolerable dose for chemotherapy.

An "individual who suffers or is at risk of undergo chemotherapy "is an individual under active treatment with a chemotherapeutic agent or an individual who has or is in risk of developing an indication for agent treatment chemotherapeutic An individual suffering from chemotherapy includes a individual currently receiving or recently receiving a dose or Chemotherapy course. Said individual may have or be in risk of developing an immunodeficiency. An individual with Chemotherapy risk includes an individual who is at point of receiving a first or subsequent doses or rounds of chemotherapy. Said individual may have an immunodeficiency or, more commonly, you are at risk of developing a immunodeficiency.

An immunodeficiency may involve a reduced number or a reduced ability to generate fully functional immune cells of at least one class, for example, CD4 + T cells, B cells, NK cells, monocytes, macrophages. An individual who has an immunodeficiency is an individual with reduced ability to have an effective immune response. Such individuals may have, for example, an immune system that is immature; an immune system that is suppressed in association with exposure to certain pharmacological agents or irradiation; or an immune system that is suppressed in association with a chromosomal defect, a hereditary or innate defect or enzyme deficiency, an antibody deficiency, a defect in the ability of T cells to process and / or present the antigen, a nutritional deficiency, an infection that directly affects the cells of the immune system (for example, HIV), or a neoplasm. These and other examples of conditions that cause an individual to be immunocompromised, can be found in Harrison's Principles of Internal Medicine , 14th Ed., Fauci AS et al., Eds., McGraw-Hill, New York, 1998.

Therefore, other types of agents chemotherapeutics that can be used according to the invention include Aminoglutethimide, Amsacrine (m-AMSA), Asparaginase, Azacitidine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Cytarabine HC1, Dactinomycin, Daunorubicin HC1, Erythropoietin, Estramustin phosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide), Ifosfamide, Leuprolide acetata (LHRH release factor analogue), Lomustine (CCNU), Mechlorethamine HC1 (nitrogen mustard), Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Mitotane (or, p'-DDD), Mitoxantrone HC1, Octreotide, Paclitaxel, Pentostatin (2'deoxicoformicin), Plicamycin, Procarbazine HC1, Semustine (methyl-CCNU), Streptozotocin, Tamoxifen citrate, Teniposide (VM-26), Thioguanine, Tiotepa, Vinblastine sulfate, and Vindesine sulfate.

The chemotherapeutic agents that currently are under development or in clinical use are shown in Table 4.

TABLE 4 Medicines in development or in the market for Cancer

9

10

eleven

12

       \ newpage
    

In one embodiment, the methods of the invention they use CpG type nucleic acids as a replacement in the use of TNF-? therapy in the treatment of cancer. In other embodiments, the methods of the invention use acids CpG type nuclei as a supplement for the use of a therapy of TNF-? in the treatment of cancer. Currently, some treatment protocols require the use of TNF-? Since the TNF-? Is produced after administration of some immunostimulatory nucleic acids, these acids nucleics can be used to generate TNF-? Endogenously.

In certain embodiments, the invention also provides methods for treating an individual who has or is at risk of developing anemia, thrombocytopenia, or neutropenia. The methods involve administering to an individual who has or is at risk of developing anemia, thrombocytopenia, or neutropenia, a CpG-type nucleic acid in an amount effective to potentiate erythropoiesis, thrombopoiesis, or neutrophil proliferation, respectively. In other words, according to these methods, the administration of the CpG type nucleic acid to the individual promotes the generation of new erythrocytes (red blood cells), platelets, or neutrophils, respectively.
mind.

Anemia is a deficiency of circulating red blood cells (erythrocytes). An "individual at risk of developing anemia", as used herein, is an individual who has any risk of exposure to an agent associated with the suppression of erythrocyte formation or its progenitors, risk of erythrocyte loss, associated with surgery or damage, or risk of developing anemia by some other mechanism that involves decreased production, accelerated destruction, and / or erythrocyte sequestration, for example, autoimmune destruction of erythrocytes, thalassemia, and renal failure. An "individual who has anemia" is an individual who has a reduced number of erythrocytes
circulating

Thrombocytopenia is a deficiency of circulating platelets An "individual at risk of developing thrombocytopenia ", as used here, is an individual who have any risk of exposure to an agent associated with the suppression of platelet formation or its parents, risk of loss of platelets, associated with surgery or damage, or risk of develop thrombocytopenia by some other mechanism that involves decreased production, accelerated destruction, and / or kidnapping of platelets, for example, autoimmune destruction of platelets. A "individual who has thrombocytopenia" is an individual who It has a small number of circulating platelets.

Neutropenia is a deficiency in neutrophils circulating An "individual at risk of developing neutropenia ", as used here, is an individual who have any risk of exposure to an agent associated with the suppression of neutrophil formation (also known as granulocytes, polymorphonuclear leukocytes, PMNs) or their parents, risk of loss of neutrophils, associated with infection and peripheral congestion or peripheral destruction after exposure to drugs that serve as immune haptens (for example, α-methyl-dopa), or risk of develop neutropenia by some other mechanism that involves decreased production, accelerated destruction, and / or kidnapping of neutrophils, for example, autoimmune destruction of neutrophils. An "individual who has neutropenia" is an individual who has a reduced number of circulating neutrophils.

CpG type nucleic acids are useful, of according to these embodiments of the invention, as prophylaxis for the treatment of an individual at risk of developing anemia, thrombocytopenia, or neutropenia. For example, nucleic acids CpG type can be administered to an individual when anticipates that the individual will be exposed to associated conditions with the development of anemia, thrombocytopenia, or neutropenia. From alternatively, CpG type nucleic acids can be administered to an individual when it is known or suspected that the individual is predisposed to develop anemia, thrombocytopenia, or neutropenia.

In addition to the use of type nucleic acids CpG and the medication for anemia, thrombocytopenia, or neutropenia for prophylactic treatment, the invention also encompasses the use of the combination of medications for the treatment of a individual who has anemia, thrombocytopenia, or neutropenia.

Anemia, thrombocytopenia, and neutropenia are frequently defined in terms of laboratory measurements, indicating a reduced hematocrit (volume percentage), a reduced platelet count (per mm3), and count of reduced neutrophils (per mm3), respectively. Methods to determine these values are well known in the state of the technique, including both automated methods and manuals The lower count limits for hematocrit and Platelets in healthy non-pregnant humans are, somehow, variables, depending on the age and sex of the individual, the method of determination, and the standards for the laboratory that performs the measurements. Generally, however, an adult human individual will says he has anemia when the hematocrit is less around from 37-40%. Similarly, generally, a adult human individual is said to have thrombocytopenia when the platelet count is below about 100,000 per mm3. Also, anemia is often measured in terms of reduced hemoglobin (g / dL) or red blood cell count (per mm3). The typical lower limits of normal values for these healthy adult humans are 12-13 g / dL and about 4.1x10 6 per mm 3, respectively. The values  corresponding for all these parameters may differ, but They are easily associable for other species.

Anemia, thrombocytopenia, and neutropenia are also frequently associated with clinical signs and symptoms in relation to its degree of severity. Anemia manifested perhaps as paleness, generalized fatigue weakness, reduced tolerance to exercise, shortening of breath with effort, fast pace of the heart, irregular heart rhythm, chest pain (angina), congestive heart failure, and headache. Thrombocytopenia,  It usually manifests in terms of spontaneous bleeding or uncontrolled, petechiae, and ease for bruises. The neutropenia is associated with infections, which include infections Notable endogenous microbial flora, and lack of inflammation.

CpG type nucleic acids can also be formulated with or administered to an individual in conjunction with an anemia medication. A "medication for anemia," as used herein, is a composition, so that it reduces the symptoms related to anemia, prevents the development of anemia, or treats existing anemia. In certain embodiments, the drug for anemia is selected from the group consisting of recombinant erythropoietin (EPO), recombinant macrophage-granulocyte colony stimulating factor (GM-CSF), granulocyte colony recombinant stimulating factor (G-CSF), IL -11 recombinant, ferrous ion, ferric ion, vitamin B12, vitamin B6, vitamin C, vitamin D, calcitriol, alfacalcidol, folate, androgen, and carnitine. In a preferred embodiment, the anemia medication is recombinant EPO. Commonly used anemia medications that are currently on the market or in development include recombinant human EPO (EPOGEN; PROCRIT), iron preparations (ferrous and ferric, CROMAGEN; FEOSOL; INFED; IROSPAN; NEFRO-FER; NEFRO-VITE ; NTFEREX; NU-IRON; SLOW FE), vitamin B12, vitamin B6, folic acid (CROMAGEN; FERRO-FOLICO; NEFRO-FER; NIFEREX), ascorbic acid, certain vitamin D metabolites (calcitriol and alfacalcidol; CALCIJEX; ROCALTROL) , androgens, anabolic steroids (ANADROL), carnitine, recombinant IL-11 (NEUMEGA), and G-CSF (NEUPOGEN). In a preferred embodiment, the anemia medication is EPO
recombinant

CpG type nucleic acids can also be formulated with or administered to an individual in conjunction with a medication for thrombocytopenia. A "medicine for thrombocytopenia ", as used here, is a composition, so that it reduces the symptoms related to the thrombocytopenia, prevents the development of thrombocytopenia, or Treat existing thrombocytopenia. In certain embodiments, the Thrombocytopenia medication is selected from the group that consists of glucocorticoid, recombinant thrombopoietin (TPO), recombinant megakaryocyte growth and development factor (MGDF), pegylated recombinant MDGF, lysophilin, Recombinant IL-11, IL-3 recombinant, recombinant IL-6, recombinant eIL-6. In a preferred embodiment, The medicine for anemia is recombinant TPO. The drugs in common use or development for the treatment of thrombocytopenia include glucocorticoids (prednisolone; prednisone; methylprednisolone; SOLUMEDROL), recombinant TPO, MGDF recombinant, pegylated recombinant MGDF, lysophilin, Recombinant IL-1, IL-3 recombinant, IL-6 recombinant, IL-11 recombinant (NEUMEGA), and recombinant G-CSF (NEUPOGEN). In a preferred embodiment, the medicament for the Thrombocytopenia is recombinant TPO.

CpG type nucleic acids can also be formulated with or administered to an individual in conjunction with a medication for neutropenia. A "medicine for neutropenia ", as used herein, is a composition, of way that reduces symptoms related to neutropenia, prevents the development of neutropenia, or treats neutropenia existing. In certain embodiments, the medication for the Tombocytopenia is selected from the group consisting of glucocorticoid, recombinant G-CSF, Recombinant GM-CSF, stimulating factor recombinant macrophage colonies (M-CSF), Recombinant IL-1, IL-3 recombinant, recombinant IL-6, immunoglobulin, androgens, EFN-?? recombinant, small mimetics of the G-CSF molecule, antagonist of the G-CSF receptor, receptor antagonists IL-3, and uteroferrin. In a preferred embodiment, The medication for neutropenia is G-CSF recombinant Commonly used medications that are currently on the market or in development for the treatment of neutropenia, include glucocorticoids (prednisolone; prednisone; methylprednisolone; SOLUMEDROL), G-CSFrecombinant (NEUPOGEN), recombinant GM-CSF (LEUKINE), Recombinant M-CSF, IL-1 recombinant, t IL-3 recombinant, Recombinant IL-6, immunoglobulin G (SANDOGLOBULIN,  IVEEGAM, GAMMAR-P, GAMIMUNE N, GAMMAGARD S / D), androgens, recombinant IFN-? (ACTIMMUNE), small mimetics of the G-CSF molecule, G-CSF receptor antagonist, antagonists IL-3 receptor, and uteroferrin. In a preferred embodiment, the medication for neutropenia is Recombinant G-CSF. Antibiotics are frequently administered in association with medications for neutropenia to treat or reduce the risk of infection.

The medications for anemia, thrombocytopenia, or neutropenia useful in combination with acids CpG type nuclei include steroids, inducers of steroids, and immunomodulators. Steroids include, but not are limited to, systematically administered corticosteroids, including methylprednisolone, prednisolone and prednisone, cortisone, and hydrocortisone. Steroid inducers include, but are not limited to adrenocorticotropic hormone (ACTH).

Corticosteroids inhibit the production of cytokines, cell adhesion activation, and activation and Migration of inflammatory cells. The secundary effects associated with systemic corticosteroids include, for example, reversible abnormalities in glucose metabolism, increased appetite, fluid retention, weight gain, mood disturbance, hypertension, peptic ulcer, and necrosis bone skeptic. Some of the secondary effectors associated with Long-term use include suppression of the adrenal axis, suppression of the growth, dermal decrease, hypertension, diabetes mellitus, Gushing syndrome, cataracts, muscle weakness, and in cases rare, damage to immune function. It is recommended that this type of Compounds are used at their lowest effective doses.

The invention also includes, still in others aspects, methods to induce innate immune activation of non-specific antigen and a broad spectrum of resistance to infectious challenges, using CpG type nucleic acids. The term "innate immune activation of the antigen does not specific ", as used herein, refers to the activation of immune cells other than B cells and, by example, it can include the activation of NK cells, T cells or other immune cells that can respond in an antigen way independent or some combination of these cells. A wide resistance spectrum to an infectious challenge is induced due to that the immune cells are in an active form and are prepared to respond to any invading component or microorganism. The cells do not have to be specifically prepared against a antigen in particular. This is particularly useful in wars. biological, and other circumstances described above as in the Travellers.

CpG type nucleic acids are effective in non-rodent vertebrates. Different nucleic acids of the CpG type can cause optimal immune stimulation, depending on the type of individual and the sequence of the nucleic acid of the CpG type. It has been found that many vertebrates, according to the invention, that are sensitive to the same class of CpG type nucleic acids, sometimes referred to as human specific CpG type nucleic acids. However, rodents respond to different nucleic acids. Therefore, the CpG type nucleic acid that causes optimal stimulation in humans generally does not have to cause optimal stimulation in a mouse, and vice versa. A CpG type nucleic acid that causes optimal stimulation in humans, however, normally causes optimal stimulation in other animals such as cows, horses, sheep, etc. A person skilled in the art can identify the optimal nucleic acid sequences useful for species of particular interest, using routine assays described herein and / or known in the state of the art, using the advice here.
supplied

CpG type nucleic acids can be administered directly to the individual or they can be co-administered with a nucleic acid delivery complex. A "nucleic acid delivery complex" would mean an associated nucleic acid molecule (for example, ionically or covalently bound; or encapsulated) to target media (for example, a molecule that results in a higher cell binding affinity target (for example, B cell surfaces and / or increase in cell adsorption by target cells)). Examples of nucleic acid delivery complexes include nucleic acids associated with a sterol (e.g., cholesterol), a lipid (e.g., cationic lipid, virosome or liposome), or a specific binding agent of the target cell (e.g., a ligand recognized by a specific target cell receptor). Preferred complexes may be stable enough in vivo to prevent significant decoupling prior to internalization by the target cell. However, the complex can be split under appropriate conditions within the cell, such that the nucleic acid is released in a functional manner. In certain embodiments, the CpG type nucleic acid may be conjugated to an antigen through a covalent bond.

The delivery vehicles or delivery mechanisms for supplying the antigen and nucleic acids to the surfaces have been described. The CpG-type nucleic acid and / or the antigen and / or other therapeutics may be administered alone (for example, in saline or buffer solutions) or using any delivery vehicle known in the state of the art. For example, the following supply vehicles have been described: Cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomas (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et al., 1998, Morein et al., 1999); Liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (eg, Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus ) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (eg, Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999 ); Microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al. , 1998, Okada et al., 1997, Ishii et al., 1997); Polymers (eg, carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); Proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); Sodium Fluoride (Hashi et al., 1998); transgenic plants (Tacket et al , 1998, Mason et al., 1998, Haq et al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al , 1998); viral type particles (Jiang et al., 1999, Leibl et al., 1998). Other delivery vehicles are known in the prior art and some additional examples are provided in the discussion of
vectors.

The language "effective amount" of a nucleic acid molecule refers to the amount necessary or sufficient to perform a biological effect. For example, an effective amount of nucleic acid containing at least one CpG type nucleic acid to treat a deficiency of the immune system could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal, or parasitic infection. An effective amount for use as an adjuvant vaccine could be that amount useful for stimulating the individual's immune response with the vaccine. An effective amount to treat asthma may be that amount useful for redirecting an immune response of type Th2, which is associated with asthma, to a response of type Th1. The amount effective for any particular application may vary depending on such factors as the disease or conditions to be treated, the particular nucleic acid being administered (for example, the number of unmethylated CpG motifs or their location in the nucleic acid) , the size of the individual, or the severity of the disease or condition. A person skilled in the art can empirically determine the effective amount of a particular oligonucleotide without the need for excessive experimentation. For any compound described herein, a therapeutically effective amount can be determined initially from animal models in vivo or in vitro by comparison with known oligonucleotides to produce an immune response.

The phrase "therapeutically effective amount" means that amount of a compound that prevents the onset of, relieves the symptoms of, or for the progression of a disorder or disease that is being treated.

The dose of the compounds described herein for mucosal or local delivery is in the typical range of about 0.1 µg to 10 µg per administration which, depending on the application, can be given daily, weekly, or monthly and any other amount of time in between. More typically, the mucosal or local doses are in the range from about 10 µg to 5 mg per administration, and more typically, from about 100 µg to 1 mg, with 2-4 administrations spaced between days or weeks. More typically, the immune stimulating dose scale is from 1 µg to 10 mg per administration, and more typically 10 µg to 1 mg with daily or weekly administrations. The doses of the compounds described herein, for parenteral delivery for the purpose of inducing an antigen-specific immune response, where the compounds are supplied with an antigen but not another therapeutic agent, are typically 5 to 10,000 times higher than the effective mucosal dose. for adjuvant vaccine or immuno-stimulant applications, and more typically 10 to 1,000 times higher, and more typically 20 to 100 times higher. The doses of the compounds described herein for parenteral delivery for the purpose of inducing an innate immune response or for increasing ADCC or for inducing an antigen-specific immune response when CpG-type nucleic acids are administered in combination with other therapeutic agents or in vehicles. Specialized delivery is in the typical range of about 0.1 µg to 10 mg per administration, depending on whether the application is given daily, weekly, or monthly and any other amount of time between means. More typically, parenteral doses for these purposes range from about 10 µg to 5 mg per administration, and more typically, from about 100 µg to 1 mg, with 2-4 administrations spaced between days or weeks. In some embodiments, however, parenteral doses for these purposes may be used on a scale of 5 to 10,000 times higher than the typical doses above.
described.

For any compound described here, the therapeutically effective amount can be determined initially from animal models. A therapeutically effective dose it can also be determined from human data to CpG oligonucleotides that have been tested in humans (have been initiated clinical trials in humans) and for other compounds that they are known exhibit similar pharmacological activities, like others mucosal adjuvants, for example, labile toxin (LT) and others antigens for vaccination purposes, for administration mucosal or local. Higher doses are required to parenteral administration The dose applied can be adjusted based on the relative bioavailability and potency of the administered compound. The dose adjustment to reach the maximum efficiency, based on the methods described above and others methods because they are well known in the state of the art, is known within the capabilities of the person skilled in the matter.

The formulations of the invention are administered in pharmaceutically acceptable solutions, which could routinely contain salt concentrations pharmaceutically acceptable buffers, preservatives, compatible vehicles, adjuvants, and, optionally, other therapeutic ingredients

For use in therapy, the effective amount of CpG type nucleic acid can be administered to an individual by any way that supplies the nucleic acid to the surface desired, for example, mucosal, systemic. The administration of the Pharmaceutical composition of the present invention can be achieved by any means known to the person skilled in the art.

Preferred routes of administration include, but not limited to, enteral (oral or any route that involves absorption from the intestinal tract), parenteral (intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, direct injection) mucosal (which includes oral, nasal, rectal, vaginal, transdermal, buccal, sublingual, pulmonary, ocular) and topical.

For oral administration, the compounds (is that is, CpG type nucleic acids, antigens and other agents therapeutic) can be easily formulated by combining the active compound (s) with vehicles pharmaceutically acceptable well known in the state of the technique. Such vehicles allow the compounds of the invention be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, suspension, suspensions and the like, for oral ingestion by an individual to be treated. Preparation is Pharmaceuticals for oral use can be obtained as an excipient solid, optionally grinding a resulting mixture, and processing the mixture of granules, after adding appropriate auxiliaries, if you want, to get pills or dragee cores. Excipients appropriate are, in particular, aggregators such as sugars, which include lactose, sucrose, mannitol, or sorbitol; preparations of cellulose such as corn starch, wheat starch, rice starch, potato starch, jelly, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). Whether desired, disintegrating agents can be added, such as polyvinyl crosslinked pyrrolidone, agar, or alginic acid or a salt thereof as sodium alginate. Optionally, oral formulations They can also be formulated in saline or buffer solutions to neutralize internal acid conditions or they can be managed without any vehicle.

Dragee cores are provided with layers adequate. For this purpose, solutions must be used sugar concentrates, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and appropriate organic solvents or solvent mixtures. The Coloring products or pigments can be added to tablets or dragee covers for identification or characterize different dose combinations of compounds assets.

The pharmaceutical preparations that can used orally include flexible capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Flexible capsules may contain the active ingredient mixed with fillers as lactose, additives such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In the soft capsules, the active compounds can be dissolved or suspended in appropriate liquids, such as fatty oils, paraffin liquid, or liquid polyethylene glycols, in addition, can be added stabilizers The microspheres formulated for oral administration They can also be used. These microspheres are well defined. in the state of the art. All formulations for oral administration may be of appropriate doses for said administration.

For oral administration, the compositions they could take the form of tablets or pills formulated from a conventional form.

For administration by inhalation, compounds for use according to the present invention, can be conveniently supplied in the form of a presentation of spray spray from pressurized packages or nebulizers, with the use of an appropriate propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other appropriate gas. In In the case of a pressurized spray, the dose unit can be determined by adding a valve to supply a quantity measure. Capsules and cartridges of, for example, jelly for their use in an inhaler or insufflator, can be formulated so that contain a powder mixture of the compound and a powder base appropriate as lactose or starch.

Compounds, when desired systematically supply, can be formulated to parenteral administration by injection, for example by bolus of continuous injection or infusion. Formulations for injection they can be presented as a unit dose, for example, in ampoules or in multi-dose containers, with a preservative added. The compositions can take such forms as suspensions, solutions or emulsions in oily vehicles or aqueous, and may contain formulation agents as agents suspensions, stabilizers and / or dispersants.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. In addition, the suspensions of the active compounds can be prepared as appropriate oleic injection suspensions. Solvents lipophilic or appropriate carriers include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Injection suspensions aqueous may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran Optionally, the suspension may also contain appropriate stabilizers or agents that increase solubility of the components, to allow the preparation of solutions highly concentrated

Alternatively, the active compounds they can be in powder form for constitution with a vehicle appropriate, for example, pyrogen-free sterile water, before use.

The compounds can also be formulated in rectal and vaginal compositions as suppositories or enemas of retention, for example, containing suppository bases conventional as cocoa cream or other glycerides.

In addition to the formulations previously described, the compounds can also be formulated as Deposit preparation Such long-acting formulations can be formulated with polymeric or hydrophobic materials appropriate (for example, as an emulsion in an acceptable oil) or ionic exchange resins, or as soluble derivatives of little quantity, for example, a soluble salt of small quantity.

Pharmaceutical compositions can also comprise appropriate solid gas or gel vehicles or excipients. Examples of such vehicles or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

The forms of suitable liquid or solid parmaceutical preparations are, for example, aqueous or saline solutions for inhalation, microencapsulated, encoded, covered with microscopic gold particles, contained in liposomes, nebulized, pellets for implantation within the skin, or dried within an object to be scratched on the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, eye drops or preparations with prolonged release of active compounds, in which preparation the excipients and additives and / or auxiliaries as disintegrants, aggregates, coating agents, swelling agents, lubricants, condiments, sweeteners or solubilizers, they are used as described above. Pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of drug delivery methods, see Langer R (1990) Science 249: 1527-1533.

CpG type nucleic acids and antigens can be administered per se (ordered) or in the form of a pharmaceutically acceptable salt. The salts, when used in medicine, may be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: acetic, sulfonic, citric, formic, hydrobromic, hydrochloric, maleic, malonic, sulfonic methane, naphthalen-2-sulfonic, nitric, phosphoric methane, p -Toluen sulfonic, salicylic, succinic, sulfuric, and tartaric. Also, said salts can be prepared as alkali metal salts or alkaline earths, such as sodium, potassium or calcium salts of the carboxylic acid group.

The appropriate buffering agents include: acetic acid and a salt (1-2% w / v), acid citric and a salt (1-3% w / v), boric acid and a salt (0.5-2.5% w / v), and phosphoric acid and a salt (0.8-2% w / v). Appropriate preservatives include Benzalkonium Chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

The pharmaceutical compositions of the invention contain an effective amount of a CpG type nucleic acid and optionally, antigens and / or other therapeutic agents optionally included in a pharmaceutically acceptable vehicle. The term "pharmaceutically acceptable vehicle" means one or more solid or liquid additives, diluents or substances encapsulants that are appropriate for administration to a human or another vertebrate animal. The term "vehicle" denotes a organic or inorganic ingredient, natural or synthetic, with which The active ingredient is combined to facilitate application. The components of the pharmaceutical compositions are also capable if combined with the compounds of the present invention, and between them, in a way that there is no interaction that could substantially impair the desired pharmaceutical efficacy.

CpG type nucleic acids useful in the invention can be supplied in mixtures with additional adjuvant (s), other therapeutic, or antigen (s). A mixture can consist of several adjuvants in addition to the nucleic acid of the CpG type or several antigens or others therapeutic

CpG type nucleic acids useful in the invention can be formulated in an amount effective to induce an immune response along with at least one adjuvant additional, another therapeutic agent, or antigen. Examples of adjuvants or antigens are provided above. With respect to others therapeutic agents that can be formulated with acids CpG type nuclei, these include, for example, a drug anticancer, antiviral agent, antibacterial agent, agent antifungal, antiparasitic agent, ulcer medication, allergy medication, asthma medication, medication for anemia, thrombocytopenia medication, medication for neutropenia, or a cytokine. Examples of each of these others therapeutic agents are provided here. Cytokines Preferred in this context include IL-2, IL-3, IL-4, IL-18, IFN-?, IFN-?, TNF-?, Flt3 ligand, G-CSF, and GM-CSF. The drugs for ulcer are useful agents in the treatment of ulcers gastrointestinal Ulcer medications include antacids, anticholinergic agents, cytoprotective agents, agents ulcer coverings / adherents, receptor antagonists histamine H2, prostaglandins, and pump inhibitors protons Non-limiting examples of specific medications for Ulcer include MAALOX, MYLANTA, ROBINUL, CYTOTEC, CARAFATE, AXED, FAMOTIDINE, PEPCID, TAGAMENT, ZANTAC, PRILOSEC, PROTONIX, and PREVACID.

There are a variety of administration routes available. The particular mode selected will depend, for of course, of the adjuvants in particular or of the antigen selected, the condition being treated in particular and the dose required for therapeutic efficacy. The methods of this invention, generally speaking, can be practiced using any mode of administration that is medically acceptable, meaning, any way that produces effective levels of an immune response without causing clinically adverse effects  not acceptable. Preferred modes of administration are discussed. down.

The compositions can be conveniently presented in unit dose form and can be prepared by any of the methods well known in the art of pharmacy. The methods may include the step of putting the compounds in association with a vehicle constituting one or more accessory ingredients In general, the compositions are prepared by uniform and intimate association of the compounds with a liquid vehicle, a finely divided solid vehicle, or both, and then, if necessary, shape the product. The units of Liquid doses are vials or ampoules. Solid dose units they are tablets, capsules, pills, troches, powders, and suppositories. For the treatment of a patient, depending on the compound activity, method of administration, purpose of immunization (ie, prophylaxis or therapeutic), nature or severity of the disorder, age and body weight of the patient, will be Different doses needed. The administration of a given dose it can be carried out both in a single administration in the form of an individual dose as in several small single doses. The multiple dose administration at specific intervals of weeks or months is normal to stimulate specific responses of antigen.

Other delivery systems may include time release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing the convenience of the individual and the physician. There are many types of release systems available and are known to those skilled in the art. These include systems based on polymers such as poly (lactide-glycolide), copolyloxalates, polycaprolactones, polyetheramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the preceding polymers containing medicaments are described in, for example, US Patent 5,075,109. The delivery systems also include non-polymeric systems which are: lipids that include sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di-, and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax covers; tablets using conventional aggregators and excipients; partially fused implants; and the like Specific examples include, but are not limited to: (a) erosion systems in which the agent of the invention is contained in a form within a matrix, such as those described in US Pat. Nos. 4,452,775,4,675,189, and 5,736,152, and (b) diffusion systems in which an active compound is impregnated in a controlled index from the polymer, as described in US Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hard object supply systems can be used, some of which are adapted for
implantation.

The present invention is illustrated by Following Examples, which are not intended to limit the scope of the invention.

       \ vskip1.000000 \ baselineskip
    

Examples Materials and methods

Oligonucleotides The modified ODN phosphorothioate (PS ODN) was purchased from Hybridon Specialty Products (Milford, MA, USA) and ARK Scientific GmbH (Darmstadt, Germany). The sequences used were (all shown in 5 'to 3' direction from left to right):

       \ vskip1.000000 \ baselineskip
    

13

14

In methylated ODN 2117, 1812, 5107, 5154, and 5155, z = 5-methylcytidine. The ODN 5246-5248 are ODN phosphorothioate structures that incorporates a 2'-methoxy modification in the sugar of deoxyribose only in cytosine residues (2'-meoxy C; 2'-OMe-C). In ODN 5280-5285, d = dSpacer. In ODN 5249, 5250, 5251, 5331, and 5387, i = inosine. In ODN 5389, m = 2,6-diaminopurine. In ODN 5384, u = uracil. In ODN 5290, w = nebularin. In ODN 5279 and 5388, 2 = 2-aminopurine. In ODN 5286, 7 = 7-deaza-guanosine. Down, be present some of these ODNs in a table form to highlight the relations between the reference ODN and the modified one (Table 5).

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

       \ vskip1.000000 \ baselineskip
    

(Table goes to page next)

fifteen

Previously, it has been shown that 2'-methoxy modifications in deoxyribose sugar decrease immunostimulation, but enhance the immunostimulatory effect of ODN with modifications at specific 2 'locations. Henry S et al. (2000) J Pharmacol Exp Ther 292: 468-79; Zhao Q et al. (1999) BioorgMed Chem Lett 9: 3453-8; Zhao Q et al. (2000) BioorgMed Chem Lett 10: 1051-4. Immune stimulation was markedly reduced with oligonucleotides containing the 2'-methoxyethoxy modifications, such that the administration, to mice, of these modified oligonucleotides did not produce splenomegaly, even at doses of 59 mg / kg. Henry S et al. (2000) J Pharmacol Exp Ther 292: 468-79. Substitution of deoxynucleosides in the flanking region of CpG-containing oligodeoxynucleoside phosphoryotioate with 2'-O-methylribonucleosides resulted in a significant decrease or increase in their immunostimulatory activities. Zhao Q et al. (1999) BioorgMed Chem Lett 9: 3453-8. The immunostimulatory activity of oligo-PS containing a CpG motif can be modulated by substitution of a single deoxynucleoside at specific sites with both 2'-O-methyl ribonucleoside and 3'-O-methyl ribonucleoside in the flanking region of the CpG motif. Zhao Q et al. (2000) BioorgMed Chem Lett 10: 1051-4.

ODNs were tested for endotoxin content (LPS) using the Limulus amoebocyte lysate test (LAL) or the LAL kinetic test (BioWhittaker, Belgium; lower limit of detection 0.1 endotoxin units (EU) / ml or 0.005EU / ml, respectively) or a method recently published by Hartmann et al. Hartmann G et al. (1999) Gene Therapy 6: 893-903. For all tests, the ODNs were diluted in TE buffer and stored at -20 ° C. All dilutions were conducted using pyrogen free reagents.

Cell preparation and cell culture. Human PMBCs were isolated from peripheral blood of healthy volunteers, were obtained by German Red Cross (Ratingen, Germany), by centrifugation in density plate-Ficoll (Histopaque-1077, Sigma, Germany) as described. Hartmann G et al. (1999) Gene Therapy 6: 893-903. The cells were suspended in RPMI1640 culture medium supplemented with 10% (v / v) heat-inactivated fetal calf serum (FCS, Sigma), 1.5 mM L-glutamine, 100 U / ml penicillin and 100 µg / ml streptomycin (all from Life Technologies, Germany). All compounds were purchased and analyzed with endotoxins. For B cell, NK cell and monocyte activation assays, PBMCs were grown in complete medium at a concentration of 2x10 6 cells / ml in 200 µl volume in 96 well plates in a humidified incubator at 37 ° C . The different DONs (see above), LPS (Sigma), IL-2, or IL-12 (R&D Systems, USA) were used as stimulus. At the indicated time points, the cells were collected by flow cytometry.

Flow cytometry. The monoclonal antibodies (mAbs) used to stain surface antigens were specific to: CD3, CD14, CD19, CD25, CD40, CD54, CD56, CD69, CD80 and CD86 (all obtained from Pharmingen / Becton Dickinson, Germany). For monocytes, Fc receptors were blocked using human IgG (Myltenyi, Germany). As described previously. Bauer M et al. (1999) Immunology 97: 699-705. Flow cytometry data of at least 1000 cells of a specific subpopulation (B cells, monocytes, NK cells, NKT cells or T cells) were acquired in FACSCalibur (Becton Dickinson) cell sorter. Data were analyzed using the CellQuest program (Becton Dickinson).

Cell Proliferation Assay The proliferating CD19 positive B cells were identified by decreased content of diester carboxyfluorescein succinimidyl diacetate (CFSE), using flow cytometry according to a recently published protocol. Hartmann G et al . (2000) J Immunol 164: 944-53.

ELISA Except where otherwise stated, PBMC (3-5x106 cells / ml) were cultured with the specified concentrations of ODN or LPS for 24 h (IL-6, TNF-?, IFN-?, And IL -10) or 8h (IL-1β) in 48-well plates in a humidified atmosphere at 37 ° C. Supernatants were collected and cytokines were measured using OPTEia ELISA kits (Pharmigen) for IL-6, TNF-?, And IFN-? Or an Eli-pair ELISA assay (Hoezel, Germany) for IL-1b e IL-10, according to the manufacturer's protocols.

TLR9 Assay . The cells used for this assay were stable transfectants (293 HEK, human embryonic kidney cells) expressing the human TLR9 receptor and containing an NF? K-luciferase genomic cassette. The cells were incubated with ODNs at 1.0 µg / ml, 6.0 µg / ml and 12.0 µg / ml for 16h. Each data was obtained in triplicate. The cells were lysed and analyzed for luciferase activity (LucLite-system, Packard). Stimulation rates were calculated in reference to luciferase activity of the medium without the addition of ODN. The activity of the ODNs is given as% of activity of the activity measured with ODN 2006, which was set at 100%. The activity of all tested ODNs was calculated as the average value of all experiments and all concentrations.
tion

       \ vskip1.000000 \ baselineskip
    

Example one

Human B cell response to CpG oligonucleotides methylated

A first group of experiments examined different ODNs, methylated and nonmethylated, for their stimulatory effect on human B cells. PBMC from several healthy male or female voluntary blood donors were incubated for 48 hours in the presence of 0.4 µg / ml, 1.0 µg / ml or 5.0 µg / ml of the following ODNs; 2006 and its 2117 methylated counterpart; 1758 (antisense ODN G3139, Genta) and its methylated counterpart 1812; 2186 (DSP30, Liang H et al. (1996) J Clin Invest 98: 1119-29) and its methylated counterpart 5107. The negative controls were similarly incubated for 48 hours in the absence of the added ODN. Then, the PBMCs were stained with mAbs for CD19 (cell B marker) and CD86 (cell B activation marker, B7-2), and the expression of CD86 on human CD19 + B cells was measured by flow cytometry. The results are shown in Fig. 1.

Fig. 1 shows that, with the exception of ODN 1812, all methylated ODNs exhibit stimulation potential B cell almost to the same degree as the corresponding sequences not methylated. The 2006 ODN at all concentrations induced a strong activation of similar B cells, in contrast to its shape methylated ODN 2117, which showed increased stimulation concentration dependent from 0.4 µg / ml to 5 µg / ml. In some experiments, ODN 2117 was only marginally very little Stimulator compared to 2006. Results were obtained equivalents for ODN 2186 with methylated ODN 5107 which has stimulatory potential Only ODN 1812 did not induce the activation of B cell at these concentrations. In another group of experiments, only an increase in CD86 expression was detected by ODN 1812 at very high concentrations of ODN (i.e. 50 µg / ml and higher), although the degree of stimulation never reached the one observed with the ODN 1758.

This unexpected high degree of stimulation of human B cell, induced by methylated CpG oligonucleotides, is investigated further and confirmed in studies of two others methylated sequences, stimulator oligonucleotides antisense 5114 (13312, Formivirsen, ISIS) (methylated: 5154) and 5116 (2302 ISIS) (methylated: 5155).

As shown in Fig. 2, both ODN antisense 5114 and 5116 exhibited significant capacity stimulant on human B cells, although it varies between different experiments, in addition, the corresponding forms methylated 5154 and 5155 also induce stimulation, although at a lower level than 5114 and 5116. These results were in agreement with the results obtained in Fig. 1 and showed that the ODN methylated immunostimulators, in fact they are compared with unmethylated sequences in their ability to induce activation of human B cells.

To exclude the possibility that observed results were due to errors during preparations or for incomplete modifications of unmethylated ODNs, tested several ODNs (2117) obtained from the same or different manufacturer to test its activity on human PBMC. The Results of these studies clearly demonstrated that all Preparations had comparable activity in human B cells.

This effect could not be attributed to contamination of ODN with bacterial endotoxins as showed in B cells that they were only poor responders to LPS

More experiments were performed to determine if these methylated ODNs were only able to enhance expression of other molecules on the surface of B cells. analogous to the experiments described, PBMC of men or women healthy blood donor volunteers incubated for 48h in presence of 0.4 µg / ml, 1.0 µg / ml or 10.0 µg / ml of the following ODN: 2006; its 2117 methylated counterpart; and 2137, the counterpart  2006 GcP. Negative controls were incubated similarly for 48 hours in the absence of added OND. After 48 hours of incubation, PBMC stained with mAb for CD19 (B cell marker) and CD80 (cell B activation marker, B7-1), CD25 (IL-2Ra), or CD54 (adhesion molecule ICAM-1). The expression of CD25, CD54, and C80 over CD19 + B cells were measured by flow cytometry. The results for CD80 and CD25 are shown in Fig. 3.

Fig. 3 demonstrates that oligonucleotides methylated, in addition to enhancing the expression of the marker of CD86 activation, induced the expression of other markers such as CD80 and CD25 on the surface of B cells. These experiments also demonstrated that methylated oligonucleotides induced the expression of CD54 on the surface of B cells human. In contrast, the ODN GpC 2137 was less stimulating than both ODN 2006 and ODN 2117, consistent with an order range of 2006 activation power> 2117> 2137 (GpC no methylated> methylated CpG> GpC).

In the B cell, ODN poliferation assays 2117 (2006 methylated) and ODN 5107 (2186 methylated) was also shown that induce human B cell proliferation comparable to those Results shown above.

       \ vskip1.000000 \ baselineskip
    

Example 2

Response of human monoliths to CpG oligonucleotides methylated

The complex effects of CpG DNA are not only due to its activation of peripheral blood B cells. Recently it was shown that these ODNs also induce the secretion of a wide variety of cytokines, by interaction with other cells, for example, monoliths, macrophages, and dendritic cells. Krieg AM (1999) Biochim Biophys Acta 1489: 107-16; Kranzer K et al. (2000) Immunology 99: 170-8; Hartmann G et al. (1999) Proc Natl Acad Sci USA 96: 9305-10; Hartmann G et al. (1999) Gene Therapy 6: 893-903. In order to investigate the role of methylated ODNs in mediating the activation of human monocytes, PBMC (2xl06 cells / ml) from several blood donors were incubated for 24 hours with 6 µg / ml of ODN 2006 , ODN 2117 (2006 methylated), ODN 2137 (GpC 2006), or 1 µg / ml of LPS. Negative controls were similarly incubated for 24 hours in the absence of added ODN or LPS. Cells were stained (after Fc-receptor blockade) with mAb for Cd14 and CD80, and CD80 expression on CD4 + monoliths was determined by flow cytometry. Fig. 4 shows two representative results of five separate experiments to compare ODN 2006, 2117, and 2137.

Similar to the results presented in Example 1 for B cells, it was found that ODN 2117 methylated induces the stimulation of human monocytes. The ODN 2006 showed the strongest activation, followed by the corresponding Methylated GpC ODN 2117 and 2137. ODN 2137 does not power (Fig. 4, donor 24) or empowered only to a low degree (Fig. 4, donor 35) the CD80 activation marker expression. LPS to a grant of 1 µg / ml, in both cases, was less stimulant than ODN 2006

       \ vskip1.000000 \ baselineskip
    

Example 3

NK cell response to CpG oligonucleotides methylated

NK cells are responsible for more than 15% of blood lymphocytes, and their main function is to recognize and kill the tumor and virus-infected cells. Roitt I, Brostoff J, and Male D, Immunology , Mosby, London, 1990. Previously it has been shown that NK cells are activated, although not directly, by proinflammatory cytokines induced by ODN to secrete IFN-γ and have a lytic activity increased for tumor cells. Ballas ZK et al. (1996) J Immunol 157: 1840-5; Takahashi T et al. (2000) J Immunol 164: 4458-64; Bohle B et al. (1999) Eur J Immunol 29: 2344-53; Krieg AM (1999) Biochim Biophys Acta 1489: 107-16. To assess the extent of activation mediated by methylated ODNs, the expression of the CD69 early activation marker on NK cells, T cells and NKT cells, upon activation, was measured in relation to exposure to methylated ODN.

PBMC (2x106 cells / ml) obtained from several blood donors were incubated for 24 hours with 6 µg / ml ODN 2006, 2117 (2006 methylated), or 2137 (GpC 2006). The controls negatives were incubated similarly for 24 hours in the absence of ODN added. As a positive control, PBMC were incubated with 100 U / ml of IL-2 together with 50 ng / ml of IL-12 To enhance the expression of CD69, the respective ODNs were added along with a submitogenic dose of IL-2 (10 U / ml). PBMC stained for CD56 (marker NK, N-CAM), CD3 (T cell marker) or CD69 (early activation marker). The expression of CD69 in the CD56 + cell population was measured by flow cytometry. The Representative results obtained from two donors are shown in Fig. 5.

As shown in both donors of Fig. 5, methylated 2117 ODN stimulated NK cells to enhance the CD69 activation marker expression, although the degree of activation was low in almost all experiments. For him On the contrary, cultivation with ODN 2137 (GpC 2006) resulted only in one very weak human NK cell stimulation. Similar to the results of Example 2 for human monocytes (Fig. 4), are observed a ranking of 2006> 2117> 2137 for the activation of NK cells

As also shown in Fig. 5, the activation of NK cells by the different ODNs was enhanced by the addition of IL-2. Takahashi T et al. (2000) J Immunol 164: 4458-64. Thus, the stimulation of NK cells by ODN 2006 marked even the strongest stimulus facilitated by the simultaneous addition of IL-2 and IL-12. The same activation force pattern was observed, 2006>2117> 2137.

More experiments investigated the activation of T cells and NKT cells in PBMC by different DONs. NKT cells have similarities with both NK and T cells. They are defined by being activated by glycolipids, exhibiting cytotoxicity against tumor cells and influencing the Th1 / Th2 pattern of the immune response. Takahashi T et al. (2000) J Immunol 164: 4458-64. PBMC (2xl06 cells / ml) were incubated with 6 µg / ml of ODN 2006, 2117, and 2137 for 24h at 37 ° C, as above. Negative controls were similarly incubated for 24 hours in the absence of added ODN. After collection, the cells were stained for CD3 (T cell marker), CD56 (NK cell marker), and CD96. The expression of CD69 on CD56 + / CD3 + NKT cells was analyzed by flow cytometry. Representative results for NKT cells are shown in Fig. 6.

Both populations of T cells and NKT cells enhanced the expression of CD69 in the culture with ODN 2006. They also showed a similar pattern of stimulation 2006> 2117> 2137. This result was in accordance with a uniform scheme of peripheral blood lymphocyte activation human by methylated and unmethylated ODN.

       \ vskip1.000000 \ baselineskip
    

Example 4

IL-6 proinflammatory cytokine secretion and TNF-? Induced by CpG oligonucleotides methylated

Proinflammatory cytokines such as IL-6 and TNF-? Play an important role in the pathogenesis of diseases. Akira S, (1990) FASEB J 4: 2860-7. Both cytokines are produced mainly by macrophages and monocytes, but also by T cells (IL-6 and TNF-?) And B cells (IL-6 only). Previous work demonstrated that CpG ODNs effectively induce the secretion of IL-6 and TNF-?. Hartmann G et al. (1999) Gene Therapy 6: 893-903. To further investigate the stimulatory effect of methylated CpG ODNs, the secretion of these cytokines was measured by PBMC after cultivation with the different ODNs.

PBMC (3x10 6 cells / ml) were obtained from several blood donors and incubated for 24 hours with 0.4, 1.0 and 6 µg / ml ODN 2006, 2117 (2006 methylated), 2137 (GpC 2006), or 1 / mug / ml of LPS as a positive control. The negative controls are similarly incubated for 24 hours in the absence of ODN or LPS added.

After 24h, the supernatants were collected and measured IL-6 and TNF-? by ELISA as described above. Representative results are shown. in Fig. 7.

The secretion of IL-6 and TNF-? Was induced by all the ODNs tested (Fig. 7A and Fig. 7B). Again, a clear activation order range 2006>2117> 2137 was observed. The same dose-response was observed for the induction of activation markers on human B cells (Fig. 7B). It is known that IL-6 mediates the differentiation and activation of B cells, as well as the secretion of antibodies (Akira S (1990) FASEB J 4: 2860-7), therefore, possibly reflecting the degree of B cell activation.

       \ vskip1.000000 \ baselineskip
    

Example 5

IL1β secretion induced by CpG oligonucleotides methylated

Of the two almost functionally equivalent forms of IL-1, IL-1? Is the predominant form in humans. This is mainly secreted by monocytes and has a wide variety of immunological functions. Bomford R and Henderson B, Interleukin-1, Inflammation and Disease , Elsevier, New York, 1989. IL-1? Plays a role in the stimulation of B, T and NK cells; participates in the conversion of Langerhans cells to professional antigen presenting dendritic cells; acts as a chemoattractant for leukocytes; and directly affects the central nervous system. Previous studies have not shown that the secretion of IL-1? Induced by CpG ODN in human PBMC, although these studies exist for murine PBMC. Chelvarajan RL et al. (1999) Eur J Immunol 29: 2808-18. The following experiments investigated the effect of CpG ODN on the secretion of human IL-1β by incubation of PBMC, isolated from healthy donor blood, with ODN 2006, 2117, and 2137 (Fig. 8).

PBMC (3x10 6 cells / ml), obtained from several blood donors were incubated for 8h with 6 µg / ml of ODN 2006, 2117, 2137, or 1 µg / ml of LPS as a positive control. Negative controls were incubated similarly for 8 hours in absence of ODN and LPS added. After 8h, the supernatants and IL-1β was measured by ELISA, as described above.

Two important results were obtained by IL-1β medium. First, the experiments showed (Fig. 8), that CpG ODNs are, in effect, potent inducers of IL-1? secretion. Second, the results demonstrated the same activation order pattern differential by non-CpG methylated and non-methylated OND 2006, 2117, and 2137, respectively.

       \ vskip1.000000 \ baselineskip
    

Example 6

Th1 and Th2 cytokine secretion induced by oligonucleotides Methylated CpG

An important feature of CpG ODNs is its potential to induce a Th1 type response in vivo. Monocytes and other cell types are directly activated to secrete Th1 cytokines, for example, IL-12.

By itself, IL-12 mediates the activation of NK cells and, thereby, the secretion of IFN-? By this cell type. Krieg AM (2000) Curr Opin Immunol 12: 35-43. A previous study showed that the secretion of IFN-γ from NK cells can be enhanced by the addition of submitogenic doses of IL-2. Iho S et al . (1999) J Immunol 163: 3642-52.

To examine the effect of methylated CpG ODN on IFN-γ secretion, PBMC were cultured (5x10 6 cells / ml), isolated from healthy donor blood, with 6 µg / ml ODN 2006, 2117, 2137, or 0.1 µg / ml LPS during 16-24h in 48-well plates in atmosphere humidified at 37 ° C. Submitogenic amounts of IL-2 (10 U / ml) to some wells containing cells and ODN, for overnight incubation. Be collected supernatants and cytokines were measured by ELISA as described above. The results are presented in the Fig. 9.

Fig. 9A illustrates that, in effect, ODN 2117 induces secretion of IFN-? in incubation of Human peripheral blood monocytes (PBMC) with this ODN. The addition of submitogenic quantities of IL-2 enhanced the secretion of IFN-? in a 5 to 10 factor (Fig. 10). Both figures show results Representative for 7 blood donors tested. But not there were big differences between the ODN, ODN 2006 induced consistent the largest amount of IFN-?, followed by ODN 2117 and 2137.

IL-10 is a cytokine that has proinflammatory properties and is thought to play a role as a regulator of the immune response induced by CpG ODN. A previous study demonstrated the induction of IL-10 after culture of murine splenocytes with CpG ODN. Redford TW et al. (1998) J Immunol 161: 3930-5.

To evaluate the possible effect of CpG ODN methylated on the secretion of IL-10, were cultured PBMC (5x10 6 cells / ml), isolated from peripheral blood of healthy blood donors, with 6 µg / ml ODN 2006, 2117, or 2137, for 16-24h in 48-well plates in humidified atmosphere at 37 ° C. Negative controls were incubated. similarly in the absence of added ODN. The ones were collected supernatants and IL-10 was measured by ELISA, as described above. The results are represented in Fig. 10.

Fig. 10 demonstrates the induction of IL-10 after incubation of human PBMC with different ODN As shown above for IFN-?, The ODN 2006 and 2117 induce higher concentrations of cytokines than ODN 2137. Consistent with the results above, the degree of immunostimulation obtained was: unmethylated ODN> ODN methylated> ODN not CpG GpC.

       \ vskip1.000000 \ baselineskip
    

Example 7

B cell response to CpI oligonucleotides

PBMC (2x10 6 cells / ml), obtained from Healthy blood donors (n = 3) were incubated for 48 hours with 0.4 µg / ml, 1.0 µg / ml, and 10.0 µg / ml of each ODN (2006, 1982, 5126, 5246, 5247, 5248, 5249, 5250, and 5251). The controls negatives were incubated similarly for 48 hours in the absence of the ODNs added. The cells were collected and stained with mAb to CD19 and CD86. CD86 activation marker expression was measured on CD19 + B cells by flow cytometry. The results Representative of 2 donors (Donor 60 and Donor 62) are shown in Fig. 11.

The results of Fig. 11 show that 2006 represents the best immunostimulatory ODN of this group, followed by ODNs not CpG 5126 and 1982. The ODNs that contain cytosine residues in its modified structure 2'-methoxy (5246-5248) not seen affected by these modifications, that is, they were so immunostimulators such as the corresponding unmodified ODN (2006, 1982, and 5126) (Fig. 11).

In related experiments, designated for investigate B cell proliferation, rather than its activation, PBMC (2x10 6 cells / ml), from 2 donors (Donor 48 and Donor 62), with the CFSE dye and then incubated with 0.4, 1.0 and 10.0 µg / ml of the same ODN panel for 5 days at 37 ° C. The negative controls were incubated similarly for 5 days in absence of added ODN. The cells were collected and stained with mAb for CD19. CD19 + B cell proliferation was measured by flow cytometry and were expressed as percentage of B cells with decrease in luminescence with the CFSE dye. The results are shown in Fig. 12.

Comparing the results for induction of B cell proliferation (Fig. 12) with activation of the cell B (Fig. 11), essentially the same can be observed differences, although in the case of PS 2'-methoxy ODN modified, the effect of the modification is not so deep. From significantly, the ODN containing inosine instead of guanine (5249-5251) was not affected by these modifications, that is, in this essay, they were so immunostimulators such as the corresponding unmodified ODN (2006, 1982, and 5126).

       \ vskip1.000000 \ baselineskip
    

Example 8

Activation of NK cells and monocytes by oligonucleotides CpI

To examine the effect on NK cells, we incubated PBMC (2x106 cells / ml) from two different donors, for 24 hours with 1.0 µg / ml and 6.0 µg / ml of each ODN (2006,1982, 5126, 5246, 5247, 5248, 5249, 5250 and 5251). The negative controls were incubated similarly for 24h in absence of added ODN. Positive controls were incubated from similar form for 24 hours with the addition of IL-2 (100 U / ml) plus IL-12 (50 ng / ml). The cells are collected and stained with CD56 cell surface markers (NK cells), CD3 (T cells) and CD69 (activation marker early). CD69 expression on NK cells was measured CD56 + / CD3 + by flow cytometry. The results are shown in Fig. 13.

To examine the effect on monocytes, PBMC (2x10 6 cells / ml), of these different donors (Donors 41, 66, and 67) were incubated for 24 hours with 1.0 µg / ml and 6.0 µg / ml from the same ODN panel. Negative controls were incubated from similarly for 24 hours in the absence of added ODN. The controls positives were incubated similarly for 24h with the addition of LPS (1 ng / ml). The cells were collected and stained with the CD14, CD19, and CD80 cell surface markers to measure the B7-1 cell surface marker expression (CD80) on CD14 + monocytes (excluding CD19 + CD14 + B cells) by flow cytometry. The results are shown in Fig. 14.

For NK cells, the ODN 2006 clearly potentiated the expression of CD69 (Fig. 13), 5126 was less active, and 1982 showed no stimulatory effect. ODN immunostimulation with modified 2'-methoxy structure cytosines (5246-5248) and guanosine changes for inosine (5249-5251) was not altered from the respective ODN Unmodified PS (2006, 1982, and 5126).

Essentially, the same graph of immunostimulatory activity for monocytes (Fig. 14). From again, the degree of activation was: ODN PS = ODN PS guanosine by inosine = ONS PS 2'-methoxy cytosine. Therefore, for all cell types tested, the ODN with the cytosines of the 2'-methoxy modified structure or those of inosine instead of guanosine, they showed the same effects as the ODN PS of reference of themselves.

       \ newpage
    

Example 9

CpI oligonucleotide-induced cytokine secretion

PBMC (5x106 cells / ml) of different donors were incubated for 20h 6.0 µg / ml ODN (2006, 1982, 5126, 5246, 5247, 5248, 5249, 5250, and 5251). Negative controls they were incubated similarly for 20h in the absence of the ODN added. Positive controls were incubated similarly for 20h in the presence of LPS (0.1 µg / ml). Supernatants cell phones were analyzed by ELISA for cytokine TNF-α proinflammatory, Th1 cytokine IFN-?, Th1 cytokine IL-10, and the cytokine IL-6 Los results are shown in Figs. 15-18.

Fig. 15 shows representative results (from donors 2 and 4) for TNF-?. TNF-? Is a cytokine that is not heavily regulated by ODN PS; typically, they can be obtained amounts between 10 and 200 pg / ml, compared to about 1000 pg / ml with LPS. In these experiments, all ODNs tested induced secretion of TNF-? (Fig. 15). For ODN 5246, 5247, and 5248 (ODN PS 2'-OMe-C) and ODN 5249, 5250, and 5251 (ODN PS inosine), these results confirm our previous observations with TNF-? levels similar to those induced by the ODN reference 2006, 1982, and 5126.

For IFN-?, These results varied depending on the donor. Representative results of 2 to 3 donors studied are shown in Fig. 16. The amounts of IFN-? In human cultures in vitro were very low, resulting in intra-experimental variations. However, again, the results demonstrated that ODNs with modified cytosines of the 2'-methoxy structure or with inosine had immunostimulatory potential. For these ODNs, the amount of IFN-γ obtained was greater than the original ODN reference.

For IL-10, they were obtained, mostly, the same results for the 2 donors studied (Fig. 17). ODN 5246-5248 and 5249-5251 ODN with cytosine structure 2'-modified methoxy and ODN PS guanosine by inosine)  induced IL-10, with the ODN containing inosine inducing larger quantities.

The results for IL-6 (Fig. 18) were consistent with those obtained for activation and B cell proliferation (Example 5). The order of degree of activation for the 4 donors studied, again it was found that was: ODN PS guanosine by inosine = PS ODN with cytosine in the 2'-methoxy modified structure.

       \ vskip1.000000 \ baselineskip
    

Example 10

Secretion of Th1 chemokine induced IP-10 by CpI oligonucleotides

In this experiment, ODN 5331, in which CpG dinucleotides of ODN 2006 were replaced by dinucleotides CpI, their ability to induce the secretion of the IP-10 chemokine induced by IFN- γ. Defrosted or fresh PBMCs and resuspended at a concentration of 5x10 6 / ml and added to 48-well plates (1 ml / well) with or without ODNs. The ODNs are they added before the cells reached a variety of final ODN concentrations, ranging from 0.4 µg / ml to 3.0 µg / ml. These cells were grown in an atmosphere humidified at 37 ° C. Culture supernatants were collected after 48h to perform ELISA. If not used immediately, the supernatants were frozen at -20 ° C until needed.

ODN 5331 (VpI) induced comparable levels of IP-10, at 3.0 mg / ml and even higher, compared with the ODN 2006.

       \ vskip1.000000 \ baselineskip
    

Example eleven

Nucleotides that include dSpacers instead of C nucleotides, G, or C and G of CpG that stimulates human B cells

The following groups of experiments examined whether the change of C, G or both nucleotides of the CpG dinucleotide by the so-called "dSpacer" (a "nucleotide" with a single serving of sugar; dSp) can affect the immunostimulatory activity of the ODNs 2006 or 5122. Therefore, these ODNs were cultured in vitro with PBMCs derived from healthy volunteers, to measure both the expression of cell expression markers on B cells and the proliferation of B cells. Surprisingly, all these ODNs 5280 (CpdSpacer by CpG), 5281 (dSpacerG by CpG), and 5282 (dSpacerpdSpacer by CpG)) exhibited at least some immunostimulatory activity. In particular, ODN 5280 showed a very high activity, almost comparable to ODN 2006 (only about 10-15% less activity). In contrast, the 5281 and 5282 ODNs induced, at most, over 50% of B-cell stimulation, compared to the 2006 ODN, and the stimulation was not enhanced by the use of higher ODN concentrations (more than 10 µg / ml) The same results were observed for reference ODN 5122 and its corresponding ODN of type CpG 5283-5285. ODN 5122 differs from ODN 2006 because it has fewer stimulatory CpG motifs.

Essentially, the same effect above is observed for the induction of B cell proliferation by these ODNs, although the difference between 2006, 5280, and the other ODNs It was more pronounced. As above, the change of G did not have the same effect that the change of C.

These results show that the C and / or G of the CpG dinucleotide can be replaced by the "dSpacer", resulting in a decrease, but not a loss, of activity immunostimulatory This preserved effect seems to be stronger. for similar ODNs of the 2006 ODN (the optimized human CpG ODN) than for the corresponding similar ODNs of ODN 5122. The ODN 2006 not only has CpG dinucleotide motifs, but also a high T. content As previously described, said sequences poly T mediates an immunostimulatory effect. Therefore, the ODN 2006 could retain part of its immunostimulatory activity after replacement of normally essential CpG dinucleotides. For other ODNs like 5122, the activity is approaching the origin (here, immunostimulation by ODN no CpG 1982) after the change of Cpg dinucleotides for a "dSpacer". Another important observation  is that the change only of the G obviously has less effect on immunostimulation that the change of the C or both nucleotides

       \ vskip1.000000 \ baselineskip
    

Example 12

ZpY oligonucleotide-induced cytokine secretion

It is these series of experiments, they were examined the 2006 ODN variants that incorporate several substitutions, different from CpI, for CpG, for its ability to induce secretion of cytokines IL-10 e IFN- γ. The OND includes ODN 5279 (CpG → Cp2, 2 = 2-aminopurine), 5280 (CpG \ rightarrowCpdSpacer (CpdSp), 5282 (CpG \ rightarrowdSppdSp), and 5290 (CpG → CpW, W = nebularin). Thawed PBMCs or fresh were resuspended at a concentration of 5x10 6 / ml and were added to 48-well plates (1 ml / well) with or without ODNs. The ODNs were added before the cells reached a variety of final ODN concentrations, varying from 0.2 µg / ml up to 0.8 µg / ml. The cells were grown in an atmosphere humidified at 37 ° C. Culture supernatants were collected after 48h to perform ELISA. If not used immediately, the supernatants were frozen at -20 ° C until needed.

The ODNs 2006, 5279, 5280, 5282, and 5290, to concentrations of 0.4 µg / ml and 0.8 µg / ml, all induced IL-10 on what was observed in the negative controls (no ODN added). ODN 5280 (CpdSp) induced similar levels of IL-10 compared to ODN 2006. In addition, ODN 5290 (CpW) also induced high comparable levels of IL-10 (700 pg / ml at 0.8 µg / ml). ODN 5279 (Cp2) was less stimulating, but still induced amounts substantial IL-10 at 0.8 µg / ml. Results similar were obtained for the secretion of IFN-?, Although ODNs 5280 and 5290 induced, in some way, smaller amounts of this Th1 cytokine than the ODN 2006 did.

       \ vskip1.000000 \ baselineskip
    

Example 13

ZpY oligonucleotide-induced B cell activation

Human PBMCs were incubated with several ZpY ODNs and the expression of an activation marker (CD86) on the B cell surface. Human PBMC, obtained from three donors, incubated with various concentrations (0.2 µg / ml at 5.0 ? / ml) of each ODN for 48h. Then the cells are collected and stained for cell marker B, CD19 and for CD86, the data was taken by flow cytometry. The data is evaluated as a stimulation index calculated in reference to samples incubated without ODNs. The 5286 ODN with a CpG change to Cp7 (7 = 7-deaza-guanosine) retained a lot of its stimulation at all concentrations examined in comparison with ODN 2006. However, ODN 5286 activated cells B much more efficiently than the non-CpG 1982 ODN, used as an ODN control. The following ODNs (also derived from the sequence of the ODN 2006) were also tested for their potential for assets B cells: CpG to UpG (ODN 5384), CpG to GpG (ODN 5385), CpG to CpC (ODN 5386), CpG to Gpl (ODN 5387), CpG to 2pG (ODN 5388) and CpG to CpM (5389, M = 2,6-diaminopurine). All these ODNs exhibited at least some immune stimulation of the cells Human immune The best ODNs were those with the changes CpG to CpC, CpM, UpG and 2pG.

       \ vskip1.000000 \ baselineskip
    

Example 14

TLR9 stimulation

The stimulation by ODN CpG depends on its great extension on activation of toll-type receiver 9 (TLR9). In this example, the activation of TLR9 transfectants by CpG ODN and CpG type ODN. As demonstrated above, the change CpG to CpI retained about 80% of the activity original (ie ODN 2006). Also, the next best ODN examined (with about 40% activity) of these transfectants, was ODN 5280 (CpG to CpdSpacer), followed by ODN 5286 (Cpg to Cp7) and ODN 5290 (CpG to CpW). These results were consistent with the comments above on the activation of Human PBMC.

       \ global \ parskip0.000000 \ baselineskip
    

<110> Coley Pharmaceutical Group, Ltd.

         \ vskip0.400000 \ baselineskip
      

<120> NUCLEIC ACIDS TYPE CpG AND METHODS OF USE OF THE SAME

         \ vskip0.400000 \ baselineskip
      

<130> C1041 / 7018WO (AWS)

         \ vskip0.400000 \ baselineskip
      

<150> US 60 / 254,341

         \ vskip0.400000 \ baselineskip
      

<151> 2000-12-08

<1S0> 36

         \ vskip0.400000 \ baselineskip
      

<170> FastSEQ for Windows Version 3.0

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

16

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 18

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (4) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (9) ... (9)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (15) ... (16)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

17

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

18

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

19

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (5)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> tn5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (21) ... (21)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

twenty

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

twenty-one

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

         \ vskip1.000000 \ baselineskip
      

22

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (5)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (7) ... (7)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (11) ... (11)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (16) ... (16)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (19)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (26) ... (27)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

2. 3

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

24

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

25

         \ vskip0.400000 \ baselineskip
      

<210> 11

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

250

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

26

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> tn5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (8) ... (8)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (10) ... (10)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (16) ... (16)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (20) ... (20)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

27

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (4)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (8) ... (8)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (12) ... (12)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (15) ... (16)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

<2 21> Modified base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (19)

         \ vskip0.400000 \ baselineskip
      

<223> m5c

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

28

         \ vskip0.400000 \ baselineskip
      

<210> 15

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (5)

         \ vskip0.400000 \ baselineskip
      

<223> cm

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (21) ... (21)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 15

29

         \ vskip0.400000 \ baselineskip
      

<210> 16

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> cm

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (8) ... (8)

         \ vskip0.400000 \ baselineskip
      

<223> cm

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (11) ... (11)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (15) ... (15)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 16

30

         \ vskip0.400000 \ baselineskip
      

<210> 17

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (5)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (19)

         \ vskip0.400000 \ baselineskip
      

<223> cm

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 17

         \ vskip1.000000 \ baselineskip
      

31

         \ vskip0.400000 \ baselineskip
      

<210> 18

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (11) ... (11)

         \ vskip0.400000 \ baselineskip
      

<223> I

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (19)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 18

         \ vskip1.000000 \ baselineskip
      

32

         \ vskip0.400000 \ baselineskip
      

<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (17) ... (18)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

         \ vskip1.000000 \ baselineskip
      

33

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 20

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (1) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (10) ... (11)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (17) ... (17)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 20

         \ vskip1.000000 \ baselineskip
      

3. 4

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 21

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (11) ... (11)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (19)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

35

         \ vskip0.400000 \ baselineskip
      

<210> 22

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 22

36

         \ vskip0.400000 \ baselineskip
      

<210> 23

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (5)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (21) ... (21)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 23

37

         \ vskip0.400000 \ baselineskip
      

<210> 24

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (21) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 24

38

         \ vskip0.400000 \ baselineskip
      

<210> 25

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (7) ... (7)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (15) ... (15)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (20) ... (20)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 25

39

         \ vskip0.400000 \ baselineskip
      

<210> 26

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (19)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 26

         \ vskip1.000000 \ baselineskip
      

40

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 27

         \ vskip0.400000 \ baselineskip
      

<211> 21

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

<22l> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (7)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (15)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (19) ... (20)

         \ vskip0.400000 \ baselineskip
      

<223> separator

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 27

         \ vskip1.000000 \ baselineskip
      

41

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 28

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> 7-deaza-guanosine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> 7-deaza-guanosine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> 7-deaza-guanosine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> 7-deaza-guanosine

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 28

42

         \ vskip0.400000 \ baselineskip
      

<210> 29

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> nebularin

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> nebularin

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> nebularin

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> nebularin

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 29

43

         \ vskip0.400000 \ baselineskip
      

<210> 30

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 30

44

         \ vskip0.400000 \ baselineskip
      

<210> 31

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 31

         \ vskip1.000000 \ baselineskip
      

Four. Five

         \ vskip0.400000 \ baselineskip
      

<210> 32

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 32

         \ vskip1.000000 \ baselineskip
      

46

         \ vskip0.400000 \ baselineskip
      

<210> 33

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 33

         \ vskip1.000000 \ baselineskip
      

47

         \ vskip0.400000 \ baselineskip
      

<210> 34

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> I

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 34

48

         \ vskip0.400000 \ baselineskip
      

<210> 35

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (2) ... (2)

<22 3> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (5) ... (5)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (13) ... (13)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (21) ... (21)

         \ vskip0.400000 \ baselineskip
      

<223> 2-aminopurine

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 35

49

         \ vskip0.400000 \ baselineskip
      

<210> 36

         \ vskip0.400000 \ baselineskip
      

<211> 24

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Synthetic oligonucleotide

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (3) ... (3)

         \ vskip0.400000 \ baselineskip
      

<223> 2,6-diaminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (6) ... (6)

         \ vskip0.400000 \ baselineskip
      

<223> 2,6-diaminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (14) ... (14)

         \ vskip0.400000 \ baselineskip
      

<223> 2,6-diaminopurine

         \ vskip0.400000 \ baselineskip
      

<221> Modified Base

         \ vskip0.400000 \ baselineskip
      

<222> (22) ... (22)

         \ vskip0.400000 \ baselineskip
      

<223> 2,6-diaminopurine

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 36

fifty

Claims (14)

1. A composition, comprising: an immunostimulatory nucleic acid that has a sequence that includes at least the following formula: 5'X_ {1} X_ {2} CGX_ {3} X_ {4} 3 ' where C is a nucleotide in which the nucleoside portion is 2'-methoxy citidine and where X 1, X 2, X 3 and X 4 are nucleotides, in a effective amount to induce a response immune 2. A composition according to claim 1, where the immunostimulatory nucleic acid:

i)

it is an isolated nucleic acid,

ii)

is between 6 and 100, or 8 and 40, nucleotides,

iii)

it has a main chain, preferably a phosphate modified main chain,

iv)

it is a synthetic nucleic acid, and / or

v)

It is at least 18 nucleotides in length and is not an antisense nucleic acid.

3. A composition according to claim 1 or 2, which further comprises a pharmaceutically acceptable vehicle. 4. A composition according to any of the claims 1-3, wherein the vehicle pharmaceutically acceptable is a release mechanism sustained. 5. A composition as claimed in the claims 2 and 3, further comprising:

i)

an antigen,

ii)

an anticancer drug, preferably, where the anticancer drug is selected from the group consisting in a monoclonal antibody, a chemotherapeutic agent, and a radiotherapeutic agent

iii)

an antiviral agent,

iv)

an antibacterial agent,

v)

an antifungal agent,

saw)

an antiparasitic agent,

vii)

an ulcer medication,

viii)

an allergy medicine,

ix)

an asthma medication,

x)

a medicine for anemia,

xi)

a medication for thrombocytopenia,

xii)

a medication for neutropenia, or

xiii)

a cytokine, preferably where the cytokine is select from the group consisting of interleukin-2  (IL-2), IL-3, IL-4. IL-18, interferon alfa (IFN-?), IFN-?, Factor of tumor necrosis alpha (TNF-?), ligand of Flt3, granulocyte colony stimulation factor (G-CSF).

6. A composition as claimed in the claims 3 and 4, wherein the composition includes at least two immunostimulatory nucleic acids that have sequences different. 7. A composition as claimed in any of claims 1-6, for use in the induction of an immune response in an individual. 8. Use of a composition as claimed in any of claims 1-6, for the manufacture of a drug to induce a response immune in an individual. 9. A use as claimed in the claim 8, wherein the individual is a dog, cat, horse, cow, pig, sheep, goat, rabbit, guinea pig, non-human primate, Chicken or fish 10. A use as claimed in the claim 8 or 9, wherein the medicament is for administration:

i)

to an individual at risk of developing a infectious disease, in an amount effective to prevent infectious disease,

ii)

to an individual with an infectious disease, in a effective amount to treat infectious disease,

iii)

to an individual at risk of developing cancer, in an effective amount to prevent cancer,

iv)

to an individual with a cancer, in an effective amount to treat cancer,

v)

to an individual at risk of developing a allergy, in an effective amount to prevent allergy,

saw)

to an individual with an allergy, in an amount effective in treating allergy,

vii)

to an individual at risk of developing asthma, in an effective amount to prevent asthma,

viii)

to an individual with asthma, in an amount effective to treat asthma,

ix)

to an individual who has or is at risk of develop an immunodeficiency, in an amount effective to enhance bone marrow proliferation in the individual, preferably when the individual who has or is at risk of developing an immunodeficiency is an individual who is undergoing or at risk of undergoing chemotherapy,

x)

to an individual who has or is at risk of develop anemia, in an amount effective to enhance the erythropoiesis in the individual,

xi)

to an individual who has or is at risk of develop thrombocytopenia, in an amount effective to potentiate thrombopoiesis in the individual,

xii)

to an individual who has or is at risk of develop neutropenia, in an amount effective to enhance the proliferation of neutrophils in the individual,

xiii)

in an amount effective to induce the production of cytokines, preferably where the cytokine is selected from group consisting of IL-1 beta (IL-1?), IL-2, IL-6, IL-12, IL-18, TNF-?, IFN-?, E IFN-?, Or

xiv)

in an amount effective to stimulate activity Natural killer cell.

11. A use as claimed in the claim 8 or 9, wherein the medicament is for administration at individual with an antigen, preferably where the antigen is select from the group consisting of an allergen, an antigen tumor, a viral antigen, a bacterial antigen, an antigen fungal, and a parasitic antigen. 12. A use as claimed in the claim 11, wherein the antigen is administered by:

i)

a mucosal route, preferably where the route Mucosal is selected from the group consisting of oral, nasal, rectal, vaginal, transdermal and ocular, or

ii)

a parenteral route, preferably where the route parenteral is selected from the group consisting of intravenous, subcutaneous, intramuscular, and direct injection.

13. A use as claimed in the claim 8 or 9, wherein the medicament is for administration at individual with an anticancer therapy, preferably where the anticancer therapy is:

i)

a monoclonal antibody specific for a cell tumor,

ii)

a chemotherapy, or

iii)

a radiotherapy

14. A use as claimed in the claim 8 or 9, wherein the medicament is for administration:

i)

on a mucosal route, preferably where the route is selected from the group you sewed in oral, nasal, rectal, vaginal, transdermal and ocular,

ii)

along a parenteral route, preferably where the parenteral route is selected from the group consisting of intravenous, subcutaneous, intramuscular, and direct injection, or

iii)

a sustained release vehicle.